Use of gc-1 in transplant related population

ABSTRACT

Methods are disclosed for the use of GC-1 (sobetirome), a prodrug thereof, or a pharmaceutically acceptable salt or prodrug thereof to increase liver regeneration and healing in liver transplant donors and recipients.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/980,645, filed May 15, 2018, which claims the benefit of U.S.Provisional Application No. 62/508,114, filed May 18, 2017. The priorapplications are incorporated herein by reference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant nos.CA204586, DK062277 and DK100287 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD

This disclosure concerns liver transplantation, specifically to the useof GC-1 (sobetirome), or prodrug thereof, or a pharmaceuticallyacceptable salt thereof, to increase liver regeneration and healing,such as in liver transplant donors and recipients.

BACKGROUND

The capacity of the liver to recover its size after resection hasallowed extensive liver resection. However, post-hepatectomy liverfailure (PHLF) remains one of the potential complications of liverresection. PHLF is defined as a post-operatively acquired deteriorationin the ability of the liver to maintain its normal functions, and is oneof the most life-threatening complications. The incidence of PHLF variesbetween 1.2% and 32%.

In PHLF there is a delicate balance between volume loss and excessgrowth, based on complex mechanisms. Several mechanisms forregeneration/regulation of liver size have been investigated, includingcytokines, growth factors, and matrix remodeling. The main concern inclinical decision-making for management of patients with insufficientliver function involves whether the liver will maintain its size andfunction after surgical resection, such as when the subject is a liverdonor. In this regard, the investigation of liver regeneration dynamicsusing factors routinely tested in the clinic represents an importantissue in practical clinical settings. However, the need remains fortreatment of PHLF.

Liver transplantation is a complex procedure that is performed at manyhealth centers in the US, as well as numerous centers in Europe andother countries. The supply of liver allografts from non-living donorsis lower than of the number of potential recipients, a reality that hasspurred the development of living donor liver transplantation. Anunderstanding of the molecular mechanisms of liver regeneration improvesthe survival rate of patients after surgical resection of large amountsof liver tissue. Liver regeneration is a well-regulated biologicalresponse to hepatocellular injury or loss involving a complex system ofinflammatory, proliferative, and metabolic mechanisms and networks; anunderstanding of these pathways and agents that can modulate them willhelp improve the survival rate of patients after surgical resection oflarge amounts of liver tissue. Methods are needed to improve graftsurvival and increase liver function in both liver transplant recipientsand liver donors.

SUMMARY

An improvement of the liver regenerative results in a reduction of theamount of liver tissue required for liver transplantation. Methods forincreasing liver regeneration also reduce the risk for the donor andenhances the growth of the transplant within the recipient.

Methods are disclosed herein for increasing proliferation ofhepatocytes. These methods include selecting a subject that is in needof increased proliferation of hepatocytes; and administering to thesubject an effective amount of a pharmaceutical composition comprisingGC-1 (sobetirome) or a pharmaceutically acceptable salt thereof, therebyincreasing hepatocyte cell number. In some embodiments, a subject isselected for treatment that does not have a liver cancer, such ashepatocellular carcinoma. In some embodiments, the subject has a liverdisease, such as cirrhosis. In further embodiments, the subject has anoverdose, such as an acetaminophen overdose.

In some embodiments, methods are disclosed for increasing hepatocytecell number in a liver transplant in a subject. These methods includeselecting a subject that is the recipient of a liver transplant; andadministering to the subject an effective amount of a pharmaceuticalcomposition comprising GC-1 (sobetirome) or a pharmaceuticallyacceptable salt thereof, thereby increasing hepatocyte cell number inthe liver transplant. In some non-limiting examples, the subject hassmall for size syndrome (SFSS). In other non-limiting examples, thesubject is the recipient of a cadaveric liver transplant. In furthernon-limiting examples, the subject is the recipient of a livertransplant from a living donor.

In additional embodiments, methods are disclosed for increasinghepatocyte cell number in a liver donor. The methods include selecting asubject that is a liver donor, wherein the subject has donated a portionof their liver, and administering to the subject an effective amount ofa pharmaceutical composition comprising GC-1 (sobetirome) or apharmaceutically acceptable salt thereof. In some non-limiting examples,the subject is an older liver donor, such as a subject of greater thanabout 45 year old, such as, but not limited to, a subject about 45-55years old.

In some embodiments, the GC-1 or the pharmaceutically acceptable saltthereof is administered intravenously to the subject, such as at a doseof 0.1 mg/kg to about 0.5 mg/kg. For example, the GC-I or thepharmaceutically acceptable salt thereof is administered at a dose of0.3 mg/kg. In other embodiments, the GC-1 or the pharmaceuticallyacceptable salt thereof is administered orally to the subject. Infurther embodiments, the GC-1 or the pharmaceutically acceptable saltthereof is administered within one day of a liver transplantation orresection procedure. In further embodiments the GC-1 or thepharmacologically acceptable salt thereof can be administered for about7, 8, 9, 10, 11, 12, 13, or 14 days.

In other embodiments, the methods include measuring the metabolicfunction of the liver in the subject and/or measuring liver size in thesubject and/or obtaining a lipid profile of the subject.

In further embodiments, the subject is human. In more embodiments, thesubject has overdosed on acetaminophen.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Thyroid hormone receptor β-agonist GC-1 induces hepatocyteproliferation in wild type mice. A. Representative photomicrographs(100×) of immunohistochemistry for BrdU and PCNA showed increasedproliferation in GC-1 treated mouse livers compared with DMSO controls.Nuclear cyclin-D1 was also increased in GC-1 treated mice. B. WesternBlot of whole liver lysates showed increased Cyclin-D1 andpSer675-β-catenin in GC-1 treated mice compared with DMSO control. C.Bar graphs shows that the difference in ALT and bilirubin (BR) levelsbetween the experimental groups and control animals were not significant(NS) and all values were within normal limits. D. Comparable normalhepatic histology in 8 days DMSO and GC-1-injected control mice (100×).

FIGS. 2A-2B. Comparison of T3 diet supplemented versus GC-1 injectedmice. A. Immunohistochemistry for BrdU and Cyclin-D1 shows increasedstaining in T3 and GC-1 treatment groups when compared with respectivecontrols. (100×). B. Bar graphs showing average number of BrdU positivehepatocyte nuclei per high power field (400×) in different conditions.Ten high power fields were counted for each group. T3 diet showed thehighest proliferation when compared with basal diet and with GC-1injections (***P<0.001). GC-1 showed increased proliferation whencompared to DMSO injected controls (***P<0.001). DMSO injected controlshad less baseline proliferation than basal diet fed animals with noother intervention (***P<0.001).

FIGS. 3A-3C. Increased hepatocyte proliferation in wild type mice fed T3supplemented (4 mg/kg) and GC-1 supplemented (5 mg/kg) diet. A. Similarhepatic histology is observed in H&E stained sections in basal diet, T3-and GC-diet fed mice as shown in representative images from all threegroups (100×). B.

Immunohistochemistry shows increased BrdU incorporation and increasednuclear Cyclin D-1 in both T3 and GC-1 diet fed groups when compared tobasal diet fed animals (100×). C. Bar graphs showing average number ofBrdU positive hepatocyte nuclei per high power field (600×) in differentconditions. Ten high power fields were counted for each group. T3 andGC-1 fed animals had significantly more BrdU-positive hepatocyte nucleithan basal diet GC-1 fed mice (***p<0.001). Interestingly, a significantdifference in hepatocyte proliferation was also evident between the T3and GC-1 diet fed mice (***p<0.001).

FIGS. 4A-4E. β-Catenin in hepatocytes is required for GC-1-inducedhepatocyte proliferation. A. Immunohistochemistry for BrdU showsdecreased staining in T3 diet fed and GC-1 injected in β-catenin-LKO ascompared to control mice. As shown in previous work, there is increasedproliferation in control mice fed T3 diet. (100×). B. Bar graphs showingaverage number of BrdU positive hepatocyte nuclei per high power field(400×) in GC-1 injected 3-catenin-LKO as compared to control mice. Totalten high power fields per group were counted. A significant differencein hepatocyte proliferation was observed between the two groups.(***p<0.001). C. Immunohistochemistry for Cycin-D1 shows increasedstaining in the livers of littermate control mice that received T3 orGC-1 as compared to notably less staining in β-catenin-LKO mice. (100×).D. Real-Time PCR shows significant decrease in mRNA expression ofCyclin-D1 in GC-1 injected β-catenin-LKO mice as compared to littermatecontrols; (one tailed T-test, *p<0.05). E. Representative Western blotsusing liver lysates from 3-catenin-LKO and controls after treatment withT3 or GC-1. As expected, β-catenin and pSer675β-catenin levels areabsent or low. Interestingly, pSer552β-catenin levels are similar towild type or slightly increased.

FIGS. 5A-5C. T3 and GC-1 induced proliferation is blunted in the absenceof redundant Wnt co-receptors LRP5-6 in hepatocytes. A. BrdU andCyclin-D1 immunohistochemistry shows decreased staining of hepatocytesin LRP5-6 LKO as compared to littermate controls in response to T3 andGC1 administration. Continued staining of smaller non-parenchymal cellsis observed in controls and LRP5-6 LKO in response to T3 and GC-1(100×). B. Bar graph shows a significant decrease in the number ofhepatocytes displaying BrdU positive nuclei per HPF (400×) in the liversections of LRP5-6 versus controls in response to T3 and GC-1(***p<0.001). C. Normalizing BrdU counts to their respective controlsshow that LRP5-6 LKO show comparably reduced percentage of hepatocytelabeling in response to T3 and GC-1.

FIGS. 6A-6F. T3 and GC-1 induce β-catenin activation via both PKA andWnt signaling. A. Representative Western blots showing comparable total3-catenin levels in liver lysates from controls and LRP5-6 administeredT3 diet for 8 days. Despite increased levels of pSer675-β-catenin andpSer552-β-catenin, decreased Cyclin-D1 was evident in LRP5-6 LKO group.GAPDH and β-Actin were loading controls. B. Representative Western blotsshowing comparable total β-catenin levels in liver lysates from controlsand LRP5-6 injected with GC-1 for 8 days. Despite comparably high levelsof pSer675-β-catenin and even greater pSer552-β-catenin levels in LRP5-6LKO liver lysates, Cyclin-D1 levels were notably lower in this group.Ponceau Red shows comparable loading in all lanes. C. Real-Time PCRshows reduced mRNA expression of Cyclin-D1 gene in the livers from GC-1injected LRP5-6 LKO as compared to controls (one tailed T-test,*p<0.05). D. Western blot shows comparable levels of TCF4 in the liverlysates of LRP5-6 LKO and littermate control livers. GAPDH shows equalloading in all lanes. E. Representative Western blot shows 8 days ofGC-1 diet fed animals leads to notably increased levels of activeβ-catenin (upper panel-darker exposure; lower panel-lighter exposure).Liver lysates from T3 or GC-1 administered β-Cat LKO show very lowlevels of active β-catenin, presumably from the non-parenchymal cells.GAPDH represents comparable loading in all lanes. F. RepresentativeWestern blot shows notable increase in active β-catenin levels in liverlysates from control littermates administered T3 or GC-1 for 8 days ascompared to basal diet. However, comparable levels of active β-cateninwere observed in LRP5-6 LKO liver lysates fed basal diet or administeredT3 or GC-1 diet for 8 days. GAPDH depicts equal loading in each lane.

FIGS. 7A-7C. Increased hepatocyte proliferation and Cyclin-D1 expressionafter partial hepatectomy in mice pre-treated with T3 or GC-1supplemented diet for 8 days. A. Immunohistochemistry for BrdU showsincreased numbers of hepatocyte staining positive after 8 days of T3 orGC-1 feeding ad libitum as compared to mice fed basal diet. The analysiswas carried out in lobes that were surgically removed at the time ofhepatectomy (Time 0). A further increase in BrdU staining was evident inregenerating livers at 24 hours in animals that were maintained on T3 orGC-1 diet post-surgery as compared to the group fed basal diet aftersurgery. (100×). B. Bar graph shows a significant increase in the numberof BrdU positive hepatocytes at 24 hours after hepatectomy in thecontrol diet fed group versus T3 diet (****p<0.0001) and control dietfed versus GC-1 diet fed group (***p<0.001). Further, significantdifference was also observed between T3- and GC-1-fed group(****p<0.0001). C. Immunohistochemistry for Cyclin-D1 shows increasednumbers of hepatocyte staining positive after 8 days of T3 or GC-1feeding ad libitum as compared to mice fed basal diet. The analysis wascarried in lobes that were surgically removed at the time of hepatectomy(Time 0). A further increase in Cyclin-D1 staining was evident inregenerating livers at 24 hours in animals that were maintained on T3 orGC-1 diet post-surgery as compared to the group fed basal diet aftersurgery (100×).

FIGS. 8A-8C. Increased hepatocyte proliferation after partialhepatectomy in mice pre-treated with T3 or GC-1 supplemented diet for 8days. A. Bar graph showing no significant (NS) differences in theaverage number of number of BrdU-positive hepatocytes per 100× field inliver sections in three independent mice on basal diet at the time ofhepatectomy (Pre-1-3) versus at 24 hours after hepatectomy in the sameanimals (Post-1-3). B. Bar graph showing significant differences(***p<0.001) in the average number of number of BrdU-positivehepatocytes per 100× field in liver sections in three independent miceon T3 diet at the time of hepatectomy (Pre-1-3) versus at 24 hours afterhepatectomy in the same animals (Post-1-3). C. Bar graph showingsignificant differences (*p<0.05; ***p<0.001) in the average number ofnumber of BrdU-positive hepatocytes per 100× field in liver sections inthree independent mice on GC-1 diet at the time of hepatectomy (Pre-1-3)versus at 24 hours after hepatectomy in the same animals (Post-1-3).

FIG. 9. Mechanism of T3/GC-1 induced β-catenin activation leading toCyclin-D1 expression and cell proliferation. In hepatocytes, T3/GC-1appears to activate β-catenin via PKA-dependent mechanism as well asthrough canonical Wnt signaling. It is likely that T3/GC-1 may induceβ-catenin activation in endothelial cells to induce cell proliferationand also may stimulate Wnt release to activate β-catenin in hepatocytesin a paracrine fashion.

FIGS. 10A-10F. GC-1 does not influence β-catenin-TCF4 activity asevident by TopFlash reporter assay in liver tumor cell lines. (A) Bargraph shows insignificant differences in TopFlash luciferase reporteractivity in Hep3B cells (have wild-type CTNNB1 gene) treated with DMSOor 70 μM GC-1. Each well for the treatment groups, is indicated by aclosed circle (DMSO) or box (GC-1).-10F. (B) Lack of TopFlash reporterresponse to GC-1 in Hep3B cells is depicted as fold-change to DMSOtreatment. (C) Bar graph shows insignificant differences in TopFlashluciferase reporter activity in Snu-389 cells (have exon-3 pointmutant-CTNNB1 gene) treated with DMSO or 70 μM GC-1. Each well for thetreatment groups, is indicated by a closed circle (DMSO) or box (GC-1).(D) Lack of TopFlash reporter response to GC-1 in Snu-398 cells isdepicted as fold-change to DMSO treatment. (E) Bar graph showsinsignificant differences in TopFlash luciferase reporter activity inHepG2 cells (have exon-3 deletion mutant-CTNNB1 gene) treated with DMSOor 70 μM GC-1. Each well for the treatment groups, is indicated by aclosed circle (DMSO) or box (GC-1). (F) Lack of TopFlash reporterresponse to GC-1 in HepG2 cells is depicted as fold-change to DMSOtreatment.

FIGS. 11A-11D: hMet-mutant-β-catenin injected mice fed GC-1 diet for 3weeks, develop lesser HCC than controls. (A) Schematic showing thetiming of GC-1 or basal diet administration and animal sacrifice inreference to the HTVI of SBTT plasmids. (B) RT-PCR using RNA isolatedfrom livers shows around 40-fold increase in gene expression ofdeiodinase after 21-days of GC-1-diet as compared to basal diet.(***p<0.001). (C) An almost significant (p=0.0506) difference in liverweight/body weight (LW/BW×100) is observed after 21-days of GC-1-diet(n=7) as compared to basal diet (n=8) suggesting a decrease in tumorburden. Individual animals in each group are indicated by a closedcircle or box. (D) Representative gross liver images from 21-days ofGC-1 versus basal diet fed animals show lesser nodularity and tumorburden in GC-1 group.

FIGS. 12A-12D. Decreased tumor size in hMet-mutant-β-catenin modelfollowing 21-days of GC-1 reflected by histology and reduced Myc-tag,without any change in cell death. (A) Representative H&E stainedsections show relatively fewer and smaller microscopic hepatic tumornodules in GC-1 treated group at 21 days (50×). (B) Representative IHCfor Myc-tag shows smaller hepatic tumor nodules at 21-days of GC-1treatment versus controls (50×). (C) Representative WB shows a modestdecrease in overall levels of Myc-tag which supports an overall lowertumor burden after 21 days of GC-1 treatment. GAPDH shows comparableloading. (D) A modestly higher number of TUNEL-positive nuclei withinthe tumor foci were evident in the control diet-fed group versusGC-1-diet-fed mice, likely due to larger size of tumor nodules (50×).

FIGS. 13A-13B. Continued proliferation in smaller tumor nodulesfollowing 21-days of GC-1 treatment as shown by numbers of cells inS-phase. (A) Scattered Ki-67 positive cells in large tumor nodules inliver sections of basal diet fed mice. While tumor nodules were smallerin GC-1 treated group, several cells continued to be Ki-67-positivecells within tumor foci (50×). (B) Quantification of Ki-67 stainingshows comparable percentage of Ki-67-positive tumor cells within foci incontrol diet and GC-1 diet fed mice. Non-tumor tissues in both groupsalso showed insignificant differences in Ki-67 positivity.

FIGS. 14A-14C. β-Catenin signaling in tumor-bearing livers remainsunaffected after 21 days of GC-1 treatment. (A) A representative sectionfrom the liver of hMet-β-catenin mice fed 21 days with GC-1 or basaldiet fed and stained for cyclin-D1 shows comparably positive stainingalthough the tumor foci were smaller after GC-1 treatment (50×). (B) Arepresentative IHC image for GS shows GS-positive tumor nodules in bothGC-1 and basal diet groups although, the foci were notably smaller inGC-1 group (50×). (C) No change in levels of total β-catenin,active-β-catenin, cyclin-D1 and marginal decrease in total GS levels byrepresentative WB, following 21-days of GC-1 treatment, suggests nochange in Wnt/β-catenin signaling. GAPDH shows comparable loading.

FIGS. 15A-15C. GC-1 treatment of hMet-mutant-β-catenin mice for 21-daysleads to marked inhibition of Met signaling. (A) A profound decrease inp-Met (Y1234/Y1235) is observed by WB analysis using whole liver lysatesfrom GC-1 treated mice versus basal diet controls. A marginal butvariable decrease in total Met was also evident in this group. GAPDHshows comparable loading. (B) Another representative WB shows lack ofp-Met (Y1234/1235) along with a dramatic decrease in downstream p-ERK1(T202) and p-ERK2 (Y204) in GC-1 treated liver lysates. Total ERK1/2levels were modestly decreased as well. GAPDH shows comparable loading.(C) A representative WB shows no change in p-AKT levels while total AKTlevels were marginally increased after GC-1 treatment. However, whiletotal STAT3 levels were relatively unaltered, a notable decrease inp-STAT3 (Y705) was clearly noticeable after 21 days of GC-1 treatment.GAPDH verified comparable protein loading.

FIGS. 16A-16D. hMet-mutant-β-catenin injected mice fed GC-1 diet for 10days, show significantly less tumors than controls. (A) Schematicshowing the timing of GC-1 or basal diet administration and animalsacrifice in reference to the HTVI of SBTT plasmids. (B) RT-PCR usingRNA isolated from livers shows around 10-fold increase in geneexpression of deiodinase after 10-days of GC-1-diet as compared to basaldiet. (***p<0.001). (C) A significant difference in liver weight/bodyweight (LW/BW×100) is observed after 10-days of GC-1-diet (n=4) ascompared to basal diet (n=4) suggesting a decrease in tumor burden.Individual animals in each group are indicated by a closed circle orbox. (**p<0.01). (D) Representative gross liver images from 10-days ofGC-1 versus basal diet fed animals show unremarkable differences in thetwo groups.

FIGS. 17A-17C. Decreased tumor volume in hMet-mutant-β-catenin modelfollowing 10 days of GC-1 diet is not due to altered cell survival orinflammation. (A) Representative H&E stained sections (upper panels)show relatively fewer and smaller microscopic tumor foci evident asnodules composed of cells with basophilia, pyknotic nuclei and greaternuclear to cytoplasmic ratio, in 10-day GC-1-diet fed group versus basaldiet group. IHC for Myc-Tag (lower panels) for 10-day GC-1 treated groupversus controls, also shows smaller and fewer tumor foci (50×). (B)Comparable TUNEL-positive nuclei within the tumor foci were evident inthe 10-day control diet-fed group versus GC-1-diet-fed mice (50×). (C)Comparable numbers of CD45-positive inflammatory cells were seen in bothbasal diet and GC-1 diet group (100×).

FIGS. 18A-18C. Decreased tumor volume in hMet-mutant-β-catenin modelfollowing 10 days of GC-1 diet is due to lower tumor cell proliferation.(A) IHC for BrdU shows a dramatic decrease in the numbers ofBrdU-positive cells after 10-days of GC-1 feeding as compared tocontrols (50×). (B) A notable decrease in the numbers of Ki-67-positivecells within tumor nodules is evident in GC1-treated group versuscontrols (50×). (C) Quantification of Ki-67 staining shows a highlysignificant (**p<0.01) decrease in percentage of Ki-67-positive tumorcells in tumor foci in 10-day GC-1 diet fed mice as compared to basaldiet group. Non-tumor tissues in both groups showed insignificantdifferences in Ki-67 positivity.

FIGS. 19A-19C: Ten days of GC-1 treatment does not impact Wnt signalingdespite lowering tumor burden. (A) Representative sections from thelivers of hMet-β-catenin mice fed 10 days with GC-1 or basal diet fedand stained for cyclin-D1 show comparably positive staining within tumorfoci although the tumor foci were smaller in the GC-1 group (50×). (B)Representative IHC for GS also shows GS-positive tumor nodules in bothGC-1 and basal diet groups although, the foci were notably smaller inGC-1 group (50×). (C) Representative WB analysis shows a marginaldecrease in overall levels of total β-catenin, cyclin-D1 and GS but notactive β-catenin, all supporting an overall lower tumor burden after 10days of GC-1 treatment but not a direct impact on Wnt/β-cateninsignaling. GAPDH shows comparable loading.

FIGS. 20A-20C. Remarkable decrease in Met-ERK and Met-STAT3 signalingfollowing 10 days of GC-1 treatment in hMet-mutant-β-catenin mice. (A) Apronounced decrease in p-Met (Y1234/Y1235) is observed and validated bytwo independent antibodies by WB analysis using whole liver lysates from10-day GC-1 treated versus basal-diet fed hMet-mutant-β-catenin mice. Amarginal decrease in total Met levels was evident in this group as well.GAPDH shows comparable loading. (B) A representative WB using totalliver lysates shows a dramatic decrease in p-ERK1 (T202) and p-ERK2(Y204) but not total ERK after 10 days of GC-1. GAPDH shows comparableloading. (C) A representative WB shows no change in total AKT or p-AKTlevels after 10 days of GC-1 treatment. Total STAT3 levels were overallreduced while p-STAT3 (Y705) levels were drastically lower after 10 daysof GC-1 treatment. GAPDH verified comparable protein loading.

FIG. 21. Schematic of disparate effect of GC-1 on β-catenin signaling innormal hepatocyte versus a liver tumor cell. Thyroid hormone receptor βagonist GC-1 induces β-catenin signaling in normal hepatocyte byactivating PKA-dependent ser675-phosphorylation (arrow) of β-catenin aswell as by Wnt-dependent mechanisms (arrow). This leads to enhancedcyclin-D1 expression and hepatocyte proliferation and may be applicablefor regenerative therapies. However, in a tumor cell, GC-1 is unable toincrease β-catenin activation (diamond short) irrespective of the CTNNB1mutational status. In addition, it dramatically inhibits Met-ERK andMet-Stat3 phosphorylation to have a profound effect on tumor burden inhMet-mutant-β-catenin mice, which represents around 10% of all humanHCC.

DETAILED DESCRIPTION

It is disclosed herein that GC-1 can be utilized post-hepatectomy, suchas in liver transplant populations, including living donors prior todonor surgery and in living donor recipients. GC-1 can also be used forthe treatment of post liver transplant cadaveric recipients with SFSS(Small for size syndrome). Without being bound by theory, the method canextend the criteria for donor livers including donor after cardiac death(DCD donors), because more livers can be utilized, thus decreasing thescarcity of organs and reducing wait list mortality.

GC-1 can also be used in high risk donors. Marginal or extended criteriadonors (ECD) are defined as those with a greater risk of initial poorfunction or graft failure and therefore an increased risk for recipientmorbidity and mortality. For example, GC-1 can be used in elderlydonors, donors with a high grade of steatosis, DCD/non-heart-beatingdonors, or split grafts. GC-1 can also be used in donors with a highDonor Risk Index (DRI), see for example,gastro_cchmc.org/calculators/donor-risk-index/, for example subjectsthat are over about 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 40 or 50years old, for example, donor that are 40 to 55 years old, such as 45 to55 years old.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as atherapeutic agent (e.g. sobetirome or a pharmaceutically acceptable saltthereof), by any effective route. Exemplary routes of administration aredescribed herein.

Hepatocyte: A cell of the main parenchymal tissue of the liver, thatmake up 70-85% of the mass of the liver. The typical hepatocyte iscubical with sides of 20-30 μm, and produces serum albumin, fibrinogen,and the prothrombin group of clotting factors (except for Factors 3 and4). Hepatocytes also synthesize lipoproteins, ceruloplasmin,transferrin, complement, and glycoproteins. A hepatocyte is a normal(non-malignant) cell.

Liver Cancer (Hepatic Cancer): Cancer that initiates from the cells ofthe liver. The most frequent liver cancer, accounting for approximately75% of all primary liver cancers, is hepatocellular carcinoma (HCC)which is formed by malignant transformation of hepatocytes. Liver cancercan also form from other structures within the liver such as the bileduct, such as cholangiocarcinoma and cholangiocellularcystadenocarcinoma. Cancers produced from muscle in the liver areleiomyosarcoma and rhabdomyosarcoma.

Liver Disease: Diseases and conditions of the liver including livercirrhosis, alcoholic and non-alcoholic fibrosis as well as to liverdisease or changes associated with obesity, diabetes and metabolicsyndrome. Other examples of liver diseases include: hepatitis, fattyliver, toxic liver failure, hepatic cirrhosis, diabetes-associated liverdisease, liver steatosis, liver fibrosis, liver cirrhosis, chronichepatitis and the like. Liver disease does not include liver cancer.

Liver regeneration: Morphologic changes in which hepatocyte growthoccurs in either a recipient or a donor of a liver transplant. Thehepatic growth generally results in an increase in hepatic function.

Liver transplantation: Partial and whole liver transplantations in whichthe liver of a donor is partially or wholly resected and partially orwholly transplanted into a recipient. In partial liver transplantation,a partial liver from a donor, corresponding to about 30-50% of thenormal liver volume of a recipient, is harvested and grafted into arecipient.

Macrovesicular Steatosis: Abnormal retention of lipids within a cell,reflecting an impairment of the normal processes of fatty acid and/ortriglyceride synthesis and elimination. Excess lipid accumulates invesicles that displace the cytoplasm. In macrovesicular steatosis, thevesicles become large enough to distort the cell's nucleus. Thecondition is not particularly detrimental to the cell in mild cases,large accumulations can disrupt cell constituents, and in severe casescells may even burst. Many different mechanisms can disrupt normal lipidmovement through the cell and cause steatosis. Those mechanisms can beclassified based on whether they result in an oversupply of lipid or afailure of lipid breakdown. Oversupply of lipid may result from, amongother conditions, obesity, insulin resistance, or alcoholism. Certaintoxins, such as alcohols, carbon tetrachloride, aspirin, and diptheriatoxin, among others, interfere with cellular machinery involved in lipidmetabolism. In addition, certain metabolic diseases are characterized bydefects in lipid metabolism. For example, in Gaucher's disease, thelysosomes fail to degrade glycolipids, resulting in steatosis.

Microvesicular Steatosis: A variant form of hepatic fat accumulationwhose histologic features contrast with the much more commonmacrovesicular steatosis. The condition was originally described inassociation with conditions sharing a number of biochemical and clinicalfeatures: acute fatty liver of pregnancy, Reye's syndrome, Jamaicanvomiting sickness, sodium valproate toxicity, high-dose tetracyclinetoxicity and certain congenital defects of urea cycle enzymes.Microvesicular steatosis has been observed in a wide variety ofconditions, including alcoholism, toxicity of several medications,hepatitis delta virus infection (primarily in South America and CentralAfrica), sudden childhood death, congenital defects of fatty acid betaoxidation, cholesterol ester storage disease, Wolman disease and Alper'ssyndrome, see. e.g., M. L. Hautekeete et al., (1990) Acta Clin. Belg.45(5):311-326.

Partial Heptatectomy or Resection: A surgical procedure in which aportion of the liver is removed. The surgeon may remove a part of theliver, such as an entire lobe, or an even larger portion of the liver.In a partial hepatectomy, the surgeon typically leaves suffienct healthyliver tissue to maintain the functions of the liver in a donor.

Pharmaceutical composition: A composition containing GC-1, or apharmaceutically acceptable salt thereof, formulated with apharmaceutically acceptable excipient, and manufactured or sold with theapproval of a governmental regulatory agency as part of a therapeuticregimen for the treatment of disease in a mammal. Pharmaceuticalcompositions can be formulated, for example, for oral administration inunit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup);for topical administration (e.g., as a cream, gel, lotion, or ointment);for intravenous administration (e.g., as a sterile solution free ofparticulate emboli and in a solvent system suitable for intravenoususe); or in any other formulation described herein.

Pharmaceutically acceptable salt: A salt of GC-1 which is, within thescope of sound medical judgment, suitable for use in contact with thetissues of humans and animals without undue toxicity, irritation,allergic response and the like and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art. For example, pharmaceutically acceptable salts are describedin: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and inPharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahland C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situduring the final isolation and purification of the compounds describedherein or separately by reacting the free carboxylic acid group with asuitable base. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, primary ammonium, secondary ammonium,tertiary ammonium, or quaternary ammonium cations, including, but notlimited to ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,ethylammonium, and the like.

Pharmaceutically acceptable excipient (pharmaceutically acceptablecarrier): Any ingredient other than GC-1, or a pharmaceuticallyacceptable salt thereof (e.g., a vehicle capable of suspending ordissolving the active compound) and having the properties of beingnontoxic and non-inflammatory in a patient. Excipients may include, forexample: antiadherents, antioxidants, binders, coatings, compressionaids, disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspensing or dispersing agents, sweeteners, or waters of hydration.Exemplary excipients include, but are not limited to: butylatedhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic),calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone,citric acid, crospovidone, cysteine, ethylcellulose, gelatin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose,magnesium stearate, maltitol, mannitol, methionine, methylcellulose,methyl paraben, microcrystalline cellulose, polyethylene glycol,polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch(corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

The pharmaceutically acceptable excipients or carriers useful for eachspecific mode of administration are described herein.

Resection: The excision of a portion or all of an organ or otherstructure. For example, liver resection refers to surgical removal of aportion of the liver and is usually performed to remove the diseasedportion of the liver, or to provide a portion of a liver for transplantinto another subject.

Small-For-Size (SFS) Liver Transplant: A surgical technique in which adonor liver is split into two or more fragments, each of which issubsequently transplanted into a different recipient. Adequate hepaticregeneration is essential for recovery of patients receiving SFStransplants, most of whom are chronically ill with severely compromisedliver function. Inadequate regeneration can result in “small-for-sizegraft syndrome,” characterized by poor bile production, intractableascites, and prolonged cholestasis, and is often associated withsurgical and septic complications.

Sobetirome (GC-1): A synthetic diarylmethane derivative that wasinvestigated clinically as a potential therapeutic forhypercholesterolemia (see U.S. Pat. No. 5,883,294, which is hereinincorporated by reference). Other names for GC-1 found in the literatureand regulatory filings include QRX-431 and GC-1.

Subject: An animal (e.g., a mammal, such as a human). A subject to betreated according to the methods described herein may be one who hasbeen diagnosed with a neurodegenerative disease involving demyelination,insufficient myelination, or under-development of a myelin sheath, e.g.,a subject diagnosed with multiple sclerosis or cerebral palsy, or one atrisk of developing the condition. Diagnosis may be performed by anymethod or technique known in the art. One skilled in the art willunderstand that a subject to be treated according to the presentdisclosure may have been subjected to standard tests or may have beenidentified, without examination, as one at risk due to the presence ofone or more risk factors associated with the disease or condition.

Therapeutically effective amount: A quantity of GC-1, or apharmaceutically acceptable salt thereof, sufficient to achieve adesired effect in a subject, or in a cell, being treated with GC-1. Theeffective amount of GC-1 depends on several factors, including, but notlimited to the subject or cells being treated, and the manner ofadministration of the therapeutic composition. In some embodiments, a“therapeutically effective amount” of GC-1, or a pharmaceuticallyacceptable salt thereof, is the amount sufficient to increase liverfunction in a subject. In other embodiments, a “therapeuticallyeffective amount” of GC-1, or a pharmaceutically acceptable saltthereof, is the amount sufficient to increase the number of liver cellsin a liver transplant donor or a liver transplant recipient. Treatingalso refers to blocking, suppressing, inhibiting, reducing, attenuating,ameliorating or reversing any or all conditions, effects or cause(s) ofthe liver injury, as well as symptoms or diseases related to the liverinjury; increasing the time between the disappearance of a condition,symptoms or effect and its reoccurrence; stabilizing an adverse symptomassociated with liver injury; or reducing, slowing, or stabilizing theprogression of a condition associated liver injury.

Transplantation of Liver: Grafting of one or more partial hepatictissue(s) or cell(s) taken or derived from another or the subject's ownliver. A transplant can be autologous (from the same subject) orallogeneic (from a different subject). Generally, a liver transplant isallogeneic. The tissue can be matched for the Major HistocompatibilityComplex (MHC) class II.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A, or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.In case of conflict, the present specification, including explanationsof terms, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Administration of GC-1(Sobetirome) or Pharmaceutical CompositionsThereof

GC-1, a prodrug thereof (see Placzek et al., Bioorg Med Chem. 2016 Nov.15; 24(22):5842-5854, incorporated herein by reference, which disclosesprodrugs of GC-1 such as ester prodrugs) and pharmaceutically acceptablesalts thereof can be administered according to any suitable route ofadministration for treatment. For example, standard routes ofadministration include intravenous, oral, parenteral, or topical routesof administration. In particular, the route of administration of GC-1, aprodrug thereof, or a pharmaceutically acceptable salt thereof can beoral (e.g., enteral, buccal, sublingual, sublabial, or by inhalation).Parenteral route of administration of GC-1, a prodrug thereof, or apharmaceutically acceptable salt thereof, can be, e.g., intra-arterial,intravenous, intraventricular, intramuscular, subcutaneous, intraspinal,intraorbital, or intracranial; these routes can also be utilized in thedisclosed methods. In some embodiments, a topical route ofadministration is utilized, such as cutaneous, intranasal, orophthalmic.

Pharmaceutical compositions comprising GC-1 have been described in theart (see, e.g., U.S. Pat. No. 5,883,294, which is herein incorporated byreference). GC-1, prodrugs thereof, and pharmaceutically acceptablesalts thereof that are to be administered intravenously or orally can beformulated as liquids, for example suspensions or emulsions, or astablets, capsules or lozenges. A liquid composition will generallyinclude a suspension or solution of GC-1, a prodrug thereof, orpharmaceutically acceptable salt in a suitable liquid carrier, forexample ethanol, glycerine, sorbitol, non-aqueous solvent such aspolyethylene glycol, oils or water, with a suspending agent,preservative, surfactant, wetting agent, flavoring or coloring agent.Alternatively, a liquid formulation can be prepared from areconstitutable powder. A liquid formulation can be prepared in dimethylsulfoxide (DMSO).

In some instances, a composition for intramuscular administrationcontains a suspension or solution of active ingredient in an oil, forexample arachis oil or sesame oil. A composition for intravenousadministration can include a sterile isotonic aqueous solutioncontaining, for example active ingredient, dextrose, sodium chloride, aco-solvent, for example polyethylene glycol and, optionally, a chelatingagent, for example ethylenediamine tetracetic acid and an anti-oxidant,for example, sodium metabisulphite. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

In some cases, for oral administration, a powder containing activecompound, suspending agent, sucrose and a sweetener can be reconstitutedwith water to form a suspension; and a syrup can be prepared from apowder containing active ingredient, sucrose and a sweetener. Acomposition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil, for example liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.GC-1, prodrugs thereof, and pharmaceutically acceptable salts thereof tobe administered parenterally can be formulated, for example, forintramuscular or intravenous administration.

GC-1, prodrugs thereof, and pharmaceutically acceptable salts thereoffor rectal administration can be formulated as suppositories. A typicalsuppository formulation will generally include active ingredient with abinding and/or lubricating agent such as a gelatin or cocoa butter orother low melting vegetable or synthetic wax or fat.

GC-1, prodrugs thereof, and pharmaceutically acceptable salts thereof tobe administered topically can be formulated as transdermal compositions.Such compositions include, for example, a backing, active compoundreservoir, a control membrane, liner and contact adhesive.

Non-limiting examples of formulations for buccal, sublingual, and/orsublabial administration may be found in U.S. Published PatentApplication No. 2012/0058962, U.S. Published Patent Application No. No.2013/0225626, U.S. Published Patent Application No. 2009/0117054, andU.S. Pat. No. 8,252,329; the disclosure of each of which is incorporatedherein by reference. For buccal, sublingual, or sublabialadministration, the compositions may take the form of tablets, lozenges,etc. formulated in a conventional manner, as described for oral dosageforms. In some embodiments, the formulation for buccal, sublingual, orsublabial administration includes one or more of taste masking agents,enhancers, complexing agents, and other described above pharmaceuticallyacceptable excipients and carriers.

Taste masking agents include, for example, taste receptor blockers,compounds which mask the chalkiness, grittiness, dryness, and/orastringent taste properties of an active compound, compounds whichreduce throat catch as well as compounds which add a flavor. A tastereceptor blocker used in the formulation of the present disclosure mayinclude Kyron T-134, a glycoprotein extract called miraculin from thefruit of the plant synsepalum dulcifcum, ethyl cellulose, hydroxypropylmethylcellulose, arginine, sodium carbonate, sodium bicarbonate,gustducin blockers and mixtures thereof. Compounds which mask thechalkiness, grittiness, dryness and/or astringent taste properties of anactive compound include those of a natural or synthetic fatty type orother flavorant such as cocoa, chocolate (e.g., mint chocolate), cocoabutter, milk fractions, vanillin butter fat, egg or egg white,peppermint oil, wintergreen oil, spearmint oil, and similar oils.Compounds which reduce throat catch include combinations of high and lowsolubility acids. For example, high solubility acids suitable for usehere include amino acids (e.g., alanine, arginine etc.), glutaric,ascorbic, malic, oxalic, tartaric, malonic, acetic, citric acids andmixtures thereof. Low solubility acids suitable for use include oleic,stearic and aspartic acids plus certain amino acids such as glutamicacid, glutamine, histidine, isoleucine, leucine, methionine,phenylalanine, serine, tryptophan, tyrosine, valine and fumaric acid.Actual amounts used will vary depending on the amount of throat catch orburn exhibited by the active used but will generally be in the range of1 to 40%. Flavoring agents include sweeteners and flavors. Examples ofsuitable sweeteners and flavors include mannitol, sorbitol, maltitol,lactitol, isomaltitol, erythritol, xylitol, sucrose, ammoniumglycyrrhizinate, mango aroma, black cherry aroma, sodium citrate,colloidal silicon dioxide, sucralose; zinc gluconate; ethyl maltitol;glycine; acesulfame-K; aspartame; saccharin; acesulfam K, neohesperidinDC, thaumatin, stevioside, fructose; xylitol; honey; honey extracts;corn syrup, golden syrup, misri, spray dried licorice root;glycerrhizine; dextrose; sodium gluconate; stevia powder; gluconodelta-lactone; ethyl vanillin; vanillin; normal and high-potencysweeteners or syrups or salts thereof and mixtures thereof. Otherexamples of appropriate flavoring agents include coffee extract, mint;lamiacea extracts; citrus extracts; almond oil; babassu oil; borage oil;blackcurrant seed oil; canola oil; castor oil; coconut oil; corn oil;cottonseed oil; evening primrose oil; grape seed oil; groundnut oil;mustard seed oil; olive oil; palm oil; palm kernel oil; peanut oil;grapeseed oil; sunflower oil; sesame oil; shark liver oil; soybean oil;hydrogenated castor oil; hydrogenated coconut oil; hydrogenated palmoil; hydrogenated soybean oil; hydrogenated vegetable oil; hydrogenatedcottonseed and castor oil; partially hydrogenated soybean oil; soy oil;glyceryl tricaproate; glyceryl tricaprylate; glyceryl tricaprate;glyceryl triundecanoate; glyceryl trilaurate; glyceryl trioleate;glyceryl trilinoleate; glyceryl trilinolenate; glyceryltricaprylate/caprate; glyceryl tricaprylate/caprate/laurate; glyceryltricaprylate/caprate/linoleate; glyceryl tricaprylate/caprate/stearate;saturated polyglycolized glycerides; linoleic glycerides;caprylic/capric glycerides; modified triglycerides; fractionatedtriglycerides; safrole, citric acid, d-limonene, malic acid, andphosphoric acid or salts and/or mixtures thereof.

Enhancers are the agents that increase membrane permeability and/orincrease the solubility of a particular active compound. Both issues canbe pivotal to the properties of the formulation. An enhancer may be achelator, a surfactant, a membrane-disrupting compound, a fatty or otheracid; a non-surfactant, such as an unsaturated cyclic urea. A chelatormay be, e.g., EDTA, citric acid, sodium salicylate, or amethoxysalicylate. A surfactant may be, e.g., sodium lauryl sulphate,polyoxyethylene, POE-9-laurylether, POE-20-cetylether, benzalkoniumchloride, 23-lauryl ether, cetylpyridinium chloride, cetyltrimethylammonium bromide, or an amphoteric or a cationic surfactant. Amembrane-disrupting compound may be, e.g., a powdered alcohol (such as,menthol) or a compound used as lipophilic enhancer. Fatty and otheracids include, e.g., oleic acid, capric acid, lauric acid, lauricacid/propylene glycol, methyloleate, yso-phosphatidylcholine, andphosphatidylcholine. Other enhancers that may be used in buccal,sublingual, and sublabial formulations of the present disclosureinclude, e.g., lysalbinic acid, glycosaminoglycans, aprotinin, azone,cyclodextrin, dextran sulfate, curcumin, menthol, polysorbate 80,sulfoxides, various alkyl glycosides,chitosan-4-thiobutylamide,chitosan-4-thiobutylamide/GSH,chitosan-cysteine, chitosan-(85% degree N-deacetylation), poly(acrylicacid)-homocysteine, polycarbophil-cysteine, polycarbophil-cysteine/GSH,chitosan-4-thioethylamide/GSH, chitosan-4-thioglycholic acid, hyaluronicacid, propanolol hydrochloride, bile salts, sodium glycocholate, sodiumdeoxycholate, sodium taurocholate, sodium glycodeoxycholate, and sodiumtaurodeoxycholate.

Buffering materials can be both used to increase solubility and enhanceadsorption of active compounds. Examples of suitable buffering materialsor antacids suitable for use herein comprise any relatively watersoluble antacid acceptable to the Food & Drug Administration, such asaluminum carbonate, aluminum hydroxide (or as aluminum hydroxide-hexitolstabilized polymer, aluminum hydroxide-magnesium hydroxide co-dried gel,aluminum hydroxide-magnesium trisilicate codried gel, aluminumhydroxide-sucrose powder hydrated), aluminum phosphate, aluminumhydroxyl carbonate, dihydroxyaluminum sodium carbonate, aluminummagnesium glycinate, dihydroxyaluminum aminoacetate, dihydroxyaluminumaminoacetic acid, bismuth aluminate, bismuth carbonate, bismuthsubcarbonate, bismuth subgallate, bismuth subnitrate, calcium carbonate,calcium phosphate, hydrated magnesium aluminate activated sulfate,magnesium aluminate, magnesium aluminosilicates, magnesium carbonate,magnesium glycinate, magnesium hydroxide, magnesium oxide, and magnesiumtrisilicate, and/or mixtures thereof. Preferred buffering materials orantacids include aluminum hydroxide, calcium carbonate, magnesiumcarbonate and mixtures thereof, as well as magnesium hydroxide. Many ofthese compounds have the advantage of also being taste masking agentsparticularly useful for addressing throat catch. The selection of theother excipients, such as permeation enhancers, disintegrants, maskingagents, binders, flavors, sweeteners and taste-masking agents, isspecifically matched to the active depending on the predeterminedpharmacokinetic profile and/or organoleptic outcome.

Liquid drug formulations suitable for use with nebulizers and liquidspray devices and electrohydrodynamic (EHD) aerosol devices willtypically include GC-1, a prodrug thereof, or a pharmaceuticallyacceptable salt thereof with a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier is aliquid, e.g., alcohol, water, polyethylene glycol, or a perfluorocarbon.Optionally, another material may be added to alter the aerosolproperties of the solution or suspension. Desirably, this material isliquid, e.g., an alcohol, glycol, polyglycol, or a fatty acid. Othermethods of formulating liquid drug solutions or suspension suitable foruse in aerosol devices are known to those of skill in the art (see,e.g., U.S. Pat. Nos. 5,112,598 and 5,556,611, each of which is hereinincorporated by reference).

Methods of Stimulating Liver Regeneration

Provided herein are methods of stimulating liver regeneration in asubject, such as, but not limited to, a liver transplant donor or aliver transplant recipient. In some embodiments, the methods stimulateliver regeneration and decrease ischemia/reperfusion injury. In someembodiments, the subject can have a partial liver resection, andoptionally can be a transplant donor. In other embodiments, the subjectcan be a recipient of a liver transplant, such as a cadaveric transplantor a transplant from a living donor. The subject can be a mammal, suchas a domestic animal or a primate. In some examples, the subject is ahuman. In some embodiments, the subject does not have liver cancer, suchas hepatocellular carcinoma.

In some embodiments, the individual has undergone a partial hepatectomyor liver resection. In some non-limiting examples, the partialhepatectomy or liver resection removed 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by mass ofthe subject's liver. In some embodiments, the subject is a liver donor.In some embodiments, the hepatectomy is anatomic, so that the lines ofresection match the limits of one or more functional segments of theliver as defined by the Couinaud classification. The subject can be aliving donor for a liver transplant. The subject can be an adult (over18 years old), or a child (under 13 years old) or a teenager (13 to 19years old). The subject can be over 20, 30, 40, 50, or 60 years old.

In further embodiments, the subject has undergone a liver transplant,and is a transplant recipient. In further embodiments the subject hasundergone a small-for-size liver transplant. In some embodiments, theindividual has undergone a liver transplant due to liver damage causedby toxic injury, traumatic injury, microvesicular steatosis, ormacrovesicular steatosis. In some non-limiting examples, the toxicinjury results from acetominophen overdose, exposure to carbontetrachloride (CCl₄), bacterial endotoxin, use or abuse of intravenousor prescription drugs, chemotherapy, excessive consumption of alcohol,or infection with hepatitis virus A, B. or C. Traumatic injury canresult from surgical resection or blunt force trauma, such as thatoccurring in an automobile accident. In certain embodiments, the methodof stimulating liver regeneration in an individual in need thereofcomprises administering to the individual a pharmaceutical compositionincluding GC-1, a prodrug thereof, or a pharmaceutically acceptable saltthereof. In some embodiments, the subject has received an extendedcriteria liver, such as, but not limited to, a liver harvested from asubject that is greater than about 45 years old, such as about 45 toabout 55 years old, such as about 45 to old 50 years old. In furtherembodiments, the subject has received a cadaveric liver. In yet otherembodiments, the subject has received a liver transplant from a livingdonor.

The dose and dosing schedule for administration of GC-1 (or a prodrugthereof of a pharmaceutically acceptable salt thereof) can vary and isdetermined in part by the clinical status of the subject, and the age,such as the weight and general health of the patient, and the route ofadministration. In some embodiments, the composition is administereddaily. In other embodiments the composition is administered more thanonce a day, such as twice a day, three time a day or four times a day.In yet other embodiments, the composition is administered once a day,every other day, every three days or once a week. In some embodiments,the drug is administered by an intravenous infusion, such as within oneday after transplantation or resection, and continued for at least 7days, such as 8, 9, 10, 11, 12, 13 or 14 days, such as for about 7 toabout 14 days. However, the IV infusion can be continued for longerperiods, such as for up to three weeks. In a liver transplant donor, andIV infusion can be administered before and/or after a resectionprocedure.

In some embodiments, GC-1, a prodrug thereof, or a pharmacologicallyacceptable salt thereof is administered intravenously, such as using aninfusion. In further embodiments, the dose of GC-1 is about 0.01 mg/kgto about 0.5 mg/kg, such as about 0.05 mg/kg to about 0.35 mg/kg, about0.01 mg/kg to about 0.30 mg/kg, or about 0.1 mg/kg to about 0.3 mg/kg.In particular examples, the dose of GC-1 (or a pharmaceuticallyacceptable salt thereof) is about 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5mg/kg. In further embodiments, the GC-1 can be administered within oneday of a surgical procedure, such as a liver resection or a livertransplantation, for example in a liver donor or in a liver recipient.The GC-1 can be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 24, 36, 48, 72 or 96 hours of the surgical procedure, and continued,as disclosed above. The GC-1, prodrug thereof, or pharmaceuticallyacceptable salt thereof, can be administered after a procedure, such as,but not limited to, in a liver transplant recipient or liver transplantdonor. The GC-1, prodrug thereof, or pharmaceutically acceptable saltthereof can be administered before a procedure, such as, but not limitedto, to a liver transplant donor or recipient.

In particular embodiments, the GC-1, a prodrug thereof, orpharmaceutically acceptable salt is administered orally, such as oncedaily, twice daily, three times daily, once every two days, once weekly,twice weekly, three times weekly, once biweekly, once monthly, or oncebimonthly. In certain embodiments, the compound is administered to thesubject once daily or twice daily. In other embodiments, the effectiveamount is more than 30 μg (e.g., more than 50 μg, such as more than 100μg). In some embodiments, the effective amount is more than 30 μg (e.g.,more than 50 μg, such as more than 100 μg) daily. In certainembodiments, the effective amount is more than 30 μg (e.g., more than 50μg, such as more than 100 μg) twice daily. In particular embodiments,the effective amount is more than 30 μg (e.g., more than 50 μg, such asmore than 100 μg) once weekly. In other embodiments, the effectiveamount is more than 30 μg (e.g., more than 50 μg, such as more than 100μg) twice weekly. In certain embodiments, the effective amount is atleast 30 μg (e.g., more than 50 μg, such as more than 100 μg) threetimes weekly. In some embodiments, the effective amount is less than 1mg (e.g., less than 500 μg, such as less than 200 μg). In furtherembodiments, the GC-1 can be administered within one day of a surgicalprocedure, such as a liver resection or a liver transplantation, forexample in a liver donor or in a liver recipient. The GC-1, prodrug orsalt can be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,24, 36, 48, 72 or 96 hours of the surgical procedure (before and/orafter the procedure). In some embodiments, the GC-1, prodrug thereof, orpharmaceutically acceptable salt thereof is given about 12 hours beforethe surgery is performed. In some embodiments, GC-1, a prodrug thereof,or a pharmaceutically acceptable salt thereof is given orally within oneday of a surgical procedure, such as a liver donation. In furtherembodiments, the GC-1 prodrug thereof, or pharmaceutically acceptablesalt thereof is administered after the surgical procedure. Thisadministration can be continued for at least 7 days, such as 8, 9, 10,11, 12, 13 or 14 days, such as for about 7 to about 14 days. However,the oral administration can be continued for longer periods, such as forup to 3, 4, 5, 6, 7 or 8 weeks. In a liver donor, administration can beprior to, and subsequent to, surgical resection of the liver. The GC-1,prodrug thereof, or pharmaceutically acceptable salt thereof, can beadministered after a procedure, such as, but not limited to, in a livertransplant recipient or liver transplant donor. The GC-1, prodrugthereof, or pharmaceutically acceptable salt thereof can be administeredbefore a procedure, such as, but not limited to, to a liver transplantdonor or recipient.

In some embodiments, the methods of the present disclosure involveadministering a unit dosage form containing from 10 μg to 100 μg ofGC-1, a prodrug thereof, or a pharmaceutically acceptable salt thereof,once, twice or three times per day orally. In some embodiments, themethods of the present disclosure involve administering a unit dosageform containing from 10 μg to 75 μg of GC-1, a prodrug thereof, or apharmaceutically acceptable salt thereof, once, twice or three times perday. In other embodiments, the methods of the present disclosure involveadministering a unit dosage form containing from 30 μg to 75 μg of GC-1,prodrug thereof, or a pharmaceutically acceptable salt thereof, once,twice or three times per day. In particular embodiments, the methods ofthe present disclosure involve administering a unit dosage formcontaining from 10 μg to 50 μg of GC-1, prodrug thereof, or apharmaceutically acceptable salt thereof, once, twice or three times perday. In yet other embodiments, the methods of the present disclosureinvolve administering a unit dosage form containing from 30 μg to 50 μgof GC-1, prodrug thereof, or a pharmaceutically acceptable salt thereof,once, twice or three times per day. In still other embodiments, themethods of the present disclosure involve administering a unit dosageform containing from 50 μg to 75 μg of GC-1, prodrug thereof, or apharmaceutically acceptable salt thereof, once, twice or three times perday. In further embodiments, the GC-1, prodrug or pharmaceuticallyacceptable salt can be administered within one day of a surgicalprocedure, such as a liver resection or a liver transplantation, forexample in a liver donor or in a liver recipient. The GC-1, prodrugthereof, or pharmaceutically acceptable salt thereof, can beadministered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48,72 or 96 hours of the surgical procedure, such as within 24 hours of asurgical procedure. The GC-1, prodrug thereof, or pharmaceuticallyacceptable salt thereof can be administered before and/or after aprocedure.

The method can include measuring liver function using a quantitativeand/or qualitative test. In some embodiments, the degree of liverimpairment is assessed using tests which evaluate structure (e.g.,biopsy), cellular permeability (e.g., transaminases) and syntheticability (e.g., albumin. bilirubin and prothrombin time) (see Jalan andHayes (1995) Aliment. Pharmacol. Ther. 9:263-270). A combination ofvarious markers for liver injury can be masured to provide an analysisfunction. Commonly used tests for liver clearance capability are:indocyanine green (ICG), galactose elimination capacity (GEC)mono-ethyl-glycine-xylidide (MEG-X), antipryine clearance, aminopyrinebreath test (ABT) and caffeine clearance. For assessment of graftfunction following transplantation, low ICG clearance and low MEG-Xformation are predictive of a poor outcome. The method can also includemeasuring the lipid profile of a subject.

Acetaminophen Overdose Treatment

In some embodiments, GC-1 or a pharmaceutical salt thereof isadministered to a subject who has taken an overdose or hepatotoxic doseacetaminophen. In some embodiments, a GC-1 or pharmaceuticallyacceptable salt thereof can that reduce the risk for livertransplantation following acetaminophen overdose. If the subject has aliver transplant subsequent to the acetaminophen overdose, then the GC-1or pharmaceutically acceptable salt thereof can be administeredfollowing the liver transplant. In further embodiments, a subject can beselected that has taken an overdose or hepatotoxic dose acetaminophen.This subject can further undergo a transplantation, but need not undergoa transplantation.

Acetaminophen is a widely used analgesic and antipyretic medication thatis generally perceived to be nontoxic. However, large or repeated dosesof acetaminophen cause profound liver injury, potentially leading toliver failure. When consumed at doses outside the therapeutic range, orin the context of altered hepatic metabolism due to alcohol, drugs suchas isoniazid, viral infections, or other concurrent medical conditions,this drug can cause significant liver damage. Acetaminophen-inducedmorbidity and mortality poses a serious clinical problem. Severe acuteliver injury due to acetaminophen overdose is a major clinical issue,and often requires liver transplantation for the survival of thepatient.

Acetaminophen overdoses are typically treated with N-acetyl-cysteine(NAC), which can prevent hepatic failure, but only if timelyadministered. When exposed to acetaminophen, the hepatocyte usesglutathione to neutralize the toxic effects of theN-acetyl-p-benzoquinoneimine metabolite of acetaminophen. The toxiceffects of this metabolite can be reversed with the addition of NAC, butthe efficacy of NAC declines precipitously as hepatocytes succumb to thetoxic effects of N-acetyl-p-benzoquinoneimine. Delayed NAC treatment foracetaminophen-induced hepatotoxicity fails, in part, because this drugfails to trigger the restoration of the critical mass of hepatocytesneeded for liver function. NAC treatment that is delayed more than eighthours after acetaminophen overdose can fail to prevent acute liverfailure. After the therapeutic window of NAC is passed, livertransplantation is often the only clinical intervention that will ensurethe survival of these patients. The GC-1 or pharmaceutically acceptablesalt thereof can be administered in combination with NAC.

Despite the evidence that the liver possesses a tremendous capacity toregenerate following hepatic injury, few biological substances canincrease hepatic cell number. GC-1 or a pharmaceutically acceptable saltthereof can be used to counteract the hepatic necrosis that followsacetaminophen-induced toxicity. In some embodiments, the GC-1 orpharmaceutically acceptable salt thereof can be administered within 1,2, 3, 4, 5, 6, 7, or 8 hours of an acetaminophen overdose. In otherembodiments, the GC-1 or pharmaceutically acceptable salt thereof isadministered more than 8, 9, 01, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 48 or 72 hours after an acetaminophen overdose. The GC-1or pharmaceutically acceptable salt thereof is administered 8, 9, 01,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48 or 72 hoursafter an acetaminophen overdose. Optionally, an effective amount of NACis also administered to the subject. Suitable doses are disclosed in thesection above. Any of the oral or intravenous infusion protocolsdisclosed for use in transplant recipients or transplant donors are ofuse in acetaminophen-induced toxicity.

If a subject that has an acetaminophen overdose requires a livertransplant, then an effective amount of GC-1, or a pharmaceuticallyacceptable salt thereof, can also be administered, as discussed above.

Liver Disease Treatment

In some embodiments, GC-1 or a pharmaceutical salt thereof isadministered to a subject who has a liver disease. In some embodiments,a GC-1 or pharmaceutically acceptable salt thereof can that reduce therisk for liver transplantation in a subject with liver disease. If thesubject has a liver transplant subsequent to the liver disease, then theGC-1 or pharmaceutically acceptable salt thereof can be administeredfollowing the liver transplant. In further embodiments, a subject can beselected that has a liver disease. This subject can further undergo atransplantation, but need not undergo a transplantation. A subject canbe selected that does not have liver cancer, such as hepatocellularcarcinoma.

Thus, a subject can be treated that has alcoholic liver cirrhosis, livercirrhosis caused by chronic infection after acute inflammation of theliver or immunological liver diseases characterized by chronicinflammation.

In some embodiments, the subject is an alcoholic or a recoveringalcoholic. The development of cirrhosis hepatitis is preceded by a stateof increasing accumulation of fat in the liver (steatosis hepatitis).This state is reversible and the liver can be normalized if consumptionof alcohol is terminated. However, if the abuse goes on then the livertissue will gradually be transformed to connective tissue which leads tobadly working liver tissue and consequently reduced function of theliver. These subjects can be treated using the methods disclosed herein.

In some embodiments, subjects are treated that have chronic liverdisease, such as liver disease wherein there are very low concentrationsof the proteins and hormones which are produced in the liver. A reducedconcentration of the protein albumin in the blood is of importance forthe development of edema in the abdominal cavity such as ascites and inthe legs caused by chronic liver disease. Subjects with liver diseasecan be treated that have a reduced capability of production ofcoagulation factors, which are important for the normal coagulation ofblood, and an increased tendency of bleeding. Thus, in some embodiments,the disclosed methods include selecting subject with one or more ofthese features.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

The capacity of the liver to regenerate has long been of interest to thescientific community. It is a focus of much research both for academicpurposes and clinical applications that could be lifesaving in cases ofend stage liver disease (Karp (2009) Am J Transplant 9, 1973-1980). Outof the midst of redundant pathways that give the liver this uniqueability, β-catenin has emerged as an important player (Monga (2014) GeneExpr 16, 51-62; Goessling et al. (2008) Dev Biol 320, 161-174).

Beta catenin is expressed throughout the adult liver. In hepatocytes, itis at the cell surface throughout the hepatic lobule. Beta cateninsignaling is crucial for the repair/regeneration of the liver especiallyfollowing surgical resection, toxic insult, infection, metabolic insult,or tumor. Beta catenin signaling is regulated through WNT signaling andWNT-independent pathways. Beta catenin can be activated via severaldifferent pathways, although the WNT signaling pathway has been the mostwidely studied. Zonation of hepatic lobule requires expression ofspecific genes in pericentral vs periportal hepatocytes for optimumhepatic function in regulating metabolism. WNT signaling might linkseveral processes linked with NASH pathogenesis including alteredglucose and lipid metabolism. WNT-β-catenin signaling is implicated inHepatic adenomas, FNH and its role in liver fibrosis is just beginningto be investigated. Prior studies show distinctive patterns of β-cateninsignaling and mutations in HCC. The various mechanisms of β-cateninactivation in HCC have a distinct impact on tumor phenotype. The overalleffects of β-catenin mutations and activation in patients with HCC, andhow these affect their prognosis are still being debated. Changes in theexpression and activity of β-catenin affect hepatic pathophysiology. Thespecific WNT proteins that modulate β-catenin activity is still notknown completely. Preliminary data shows that activation of β-catenincould induce liver regeneration. Triiodothyronine can activate β-cateninin rodents.

Triiodothyronine (T3) induces hepatocyte proliferation in rodents. T3has mitogenic effects through in large part via activation of β-cateninsignaling. The hepatocytes mostly express T3 hormone receptor β (TRβ).GC-1 is a selective TRβ agonists which has β-catenin-dependenthepatocyte proliferation effect. A study shows that TRβ-selectiveagonists like GC-1 can induce hepatocyte proliferation through β-cateninactivation in rodents via Wnt-dependent and Wnt-independent mechanismsand confer a regenerative advantage following surgical resection. Thedevelopment of alternative methods of treatment of liver disease andregeneration is in great demand. The emerging field of regenerativemedicine offers novel approaches. Regenerative response to partialhepatectomy involves numerous coordinated events occurring at themolecular, cellular, biochemical and tissue levels. Hepatocytehypertrophy starts within hours after partial hepatectomy and isfollowed by hepatocyte hyperplasia. Restoration after injury involvesrestitution of all functions of normal liver including synthetic andmetabolic function. GC-1 can accelerate liver regeneration which couldbe potentiate the normal physiological process.

Examples Example 1 Materials and Methods

Animals. All studies on mice were performed in strict accordance withGuidelines. Eight-week old male C57B/6 male mice (Jackson/Charles River)were maintained on a standard laboratory basal diet (Test Diet) (n=4),or fed a T3-supplemented diet (4 mg/Kg of diet, Sigma Chemicals, St.Louis Mo.) (n=4). GC-1 was administered to the eight week-old maleC57BL/6 male mice either via GC-1-supplemented diet (5 mg/Kg of diet,Medchem Express) for 8 days (n=4) or via daily intraperitonealinjections (IP) of GC-1 dissolved in DMSO (0.3 mg/kg/dose) for 8 days(n=7). As a control group for injections, DMSO alone was injected dailyintraperitoneal (IP) for 8 days (n=5). Mice were sacrificed 24 hoursafter the last injection.

Eight to 10 week old male hepatocyte specific β-catenin knockout mice(β-cat-LKO) or sex-matched littermate controls obtained from breedinghomozygous floxed β-catenin mice and albumin-cre transgenic mice (asdescribed elsewhere (Tan et al. (2006) Gastroenterology 131, 1561-1572))were given 8 daily IP injections of GC-1 (n=4) or DMSO (n=4) andharvested around 24 hours after the last injection.

Eight to 10 week-old LRP5-6-LKO were obtained from breeding homozygousLRP5-6 double floxed mice with albumin-cre transgenic mice as describedpreviously (Yang et al. (2014) Hepatology 60, 964-976). LRP5-6-LKO orsex matched littermate controls were given either T3-supplemented diet(n=4), basal diet (n=3), IP GC-1 (n=3) or IP DMSO (n=3). Mice weresacrificed at 24 hours after the last IP injection.

To label dividing hepatocytes, BrdU (5-bromodeoxyuridine) dissolved indrinking water (1 mg/ml) was given to all animals throughout theexperiment period. The animals were given food and water ad libitum witha 12-hour light/dark daily cycle.

Partial Hepaectomy. After 7 days of receiving T3 supplemented diet (n=3)or GC-1 diet (n=3) or standard basal mouse chow (n=3), C57BL/6 male miceunderwent partial hepatectomy as described previously (Tan et al. (2006)Gastroenterology 131, 1561-1572). Anesthesia was provided with inhaledIsoflurane. The animals were sacrificed 24 hours post hepatectomy, afterIsoflurane anesthesia and cervical dislocation. Serum and liver tissuewere harvested for further processing.

Immunohistochemistry. Four micron liver sections were analyzed byimmunohistochemistry for β-Catenin (BD Biosciences, San Jose, Calif.),Cyclin-D1 (Thermo-Scientific, Fremont, Calif.) and PCNA (Santa CruzBiotechnology, Dallas, Tex.). Briefly, formalin-fixed sections weredeparaffinized in graded xylene and alcohol. Endogenous peroxidase wasinactivated using 3% hydrogen peroxide (Sigma). For S-Catenin, Cyclin-D1staining, slides were microwaved in Citrate buffer followed by blockingwith Superblock for 10 minutes. Sections were then incubated withsecondary anti-mouse (for β-catenin) or anti-rabbit (for Cyclin-D1)horseradish peroxidase-conjugated antibody for 30 minutes.

For PCNA, slides were microwaved in zinc sulfate for 12 minutes,followed by blocking with Superblock for 20 minutes. Sections wereincubated with PCNA antibody (Santa Cruz) diluted 1:4,000 in phosphatebuffered saline (PBS) for 60 minutes. Slides then incubated with horseanti-mouse secondary antibody (1:700 dilution) for 30 minutes. ABC wasapplied for 30 minutes. DAB kit was then applied. Slides were thendehydrated, coverslips placed, and allowed to dry.

Bromodeoxyuridine (BrdU) was stained with mouse antibody (BectonDickinson) as previously described (Fanti et al. (2014) Hepatology 59,2309-2320). Briefly, tissue sections were deparaffinized, exposed to0.3% hydrogen peroxide in deionized water for 10 minutes to blockendogenous peroxidase, treated with 2N HCl, incubated with trypsin 0.1%for 20 minutes and then with normal goat serum for 20 minutes at roomtemperature. Sections were then incubated overnight in cold room withanti-BrdU monoclonal antibody, followed by biotinylated goat anti-mouseIgG. DAB kit was then applied, and sections were counterstained withhematoxylin. A small segment of intestine was included from each mouseas a positive control for the staining. For quantification of BrdUstaining, at least 10 high power fields (400× or 600×) fields each fromat least three biological replicates were counted as indicated in thefigures. Statistical analysis was performed as described below.

Protein Extraction and Western Blot Analysis. Protein extraction fromfrozen liver tissue and western blot analysis were performed aspreviously described (Fanti et al., (2014) Hepatology 59, 2309-2320).For protein extraction, a small amount of frozen liver tissue wasobtained (over dry ice) and was homogenized using RIPA buffer withprotease and phosphatase inhibitor cocktail (Sigma, St. Louis, Mo.).Samples were transferred to a 1.5 ml tube on ice, and spun in cold roomcentrifuge at 14,000 RPM for 5 minutes. Pellet was then discarded andsample was aliquoted and stored at −80 C for use. Fifty micrograms ofthe proteins were resolved by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis analysis, and transferred to immobilon-polyvinylidenedifluoride membranes.

The primary antibodies used were against β-catenin (BD Biosciences);pSer675-β-catenin, pSer552-β-catenin, Active-β-catenin (Cell-Signaling),Cyclin-D1 (Thermo-Scientific), and β-Actin (Sigma). Dilutions of primaryantibody were done according to manufacturer recommendations (1:1000 forall except Cyclin-D1 which was 1:200). Horseradish peroxidase-conjugatedsecondary antibodies used were goat anti-mouse (1:20,000) and goatanti-rabbit (1:10,000) (Millipore). The proteins were detected bySuper-Signal West Pico Chemiluminescense Substrate (Thermo Scientific,Rockford Ill.) and visualized by autoradiography.

Real Time Polymerase Chain Reaction (PCR). Total RNA was extracted byhomogenizing frozen liver tissues in Trizol reagent (Invitrogen,Carlsbad, Calif.) from GC-1-injected J-Cat LKO (n=4) and littermatecontrol mice (n=4); and GC-1-injected LRP5-6 LKO (n=3) and littermatecontrol mice (n=3). Two micrograms of total RNA from each sample wasreverse-transcribed after DNAse treatment using Super Script III firststrand kit (Invitrogen). Real-time PCR was performed on an ABI Prism7300 Sequence Detection System (applied Biosystems, Foster City, Calif.)using SYBR Green. For Cyclin-D1, values were normalized to GAPDH.

Statistics. Data are presented as mean±SD or SE as indicated. Allstatistics were performed using Prism 6 software (GraphPad) for Mac OSX. Comparisons between various groups were performed by Student t test(for two groups) or ordinary one-way ANOVA for multiple comparisons.Analysis for significance was done by Tukey's multiple comparisons test.P values of <0.05 (*), <0.01 (**), <0.001 (***) and <0.0001 (****) wereconsidered significant throughout the study.

Example 2 GC-1 Treatment Induces Hepatocyte Proliferation, β-CateninActivation, and Cyclin-D1 Expression in Wild Type Mice without Evidenceof any Liver Injury

T3 hormone was previously shown to exert a strong mitogenic effect overhepatocytes in rats and mice, and this action has been shown to requireβ-catenin activation (Fanti et al. (2014) Hepatology 59, 2309-2320). Todetermine if TRβ-agonist GC-1 also causes hepatocyte proliferation, anexperiment with normal mice was first performed. Two to three-month oldC57BL/6 mice received daily intraperitoneal (IP) injections with GC-1(0.3 mg/Kg/dose) dissolved in dimethyl sulfoxide DMSO, or DMSO alone ascontrol. The results showed increased number of PCNA-positivehepatocytes as well as BrdU incorporation in GC-1 group showing anincrease in hepatocyte proliferation in this group as compared to theDMSO control (FIG. 1A). Immunohistochemistry for cyclin-D1 also showedmany positive hepatocytes in GC-1 but not DMSO control (FIG. 1A).Western Blot analysis using whole cell lysates from the livers from thetwo groups showed increased β-Ser675-β-catenin and cyclin-D1 levels inGC-1 treated group while total β-catenin levels did not show muchdifference (FIG. 1B). Serum analysis from the two groups of mice showedinsignificant differences in serum ALT or total bilirubin, whichremained normal (FIG. 1C). Further, normal hepatic histology comparableto DMSO injected control was evident in GC-1 group as shown inrepresentative H&E staining (FIG. 1D). Thus GC-1 administration by dailyIP injections induces hepatocyte proliferation through activation ofβ-catenin and increased cyclin-D1 without any untoward biochemical orhistological consequences.

Example 3 GC-1 Induces Hepatocyte Proliferation

An initial comparison of T3 supplemented diet versus IP GC-1 for 8 dayswith ad libitum access to BrdU containing drinking water showed anoverall fewer number of BrdU-positive hepatocytes indicating lesserproliferation in the GC-1 treated group (FIG. 2A). This went along withnotably increased cyclin-D1 staining in hepatocytes after T3 and GC-1 ascompared to their respective controls, although greater numbers ofpositive cells were evident in the T3 group (FIG. 2A). WhenBrdU-positive hepatocytes were carefully counted in T3-fed andGC-1-injected groups, the difference between the two groups wasstatistically significant (p<0.001) (FIG. 2B). Interestingly however, adecrease in the number of BrdU labeled hepatocyte nuclei was alsoobserved in the DMSO injected mice when compared to mice on basal dietand this difference was also statistically significant (FIG. 2B).

Differences between the mode of drug delivery plus the finding of lowerbasal level of proliferating cells in the DMSO group prompted us tocompare hepatocyte proliferation in mice administered T3 and GC-1 viasupplemented diets. Normal male mice were fed regular rodent chow,T3-supplemented (4 mg/Kg of diet) chow, or GC-1-supplemented (5 mg/Kg ofdiet) chow for 8 days. As with GC-1 IP injection, no histologicalchanges in the livers were observed after GC-1 or T3 feeding as comparedto the control basal diet (FIG. 3A). Mice fed T3 or GC-1 supplementeddiets showed increased BrdU incorporation when compared to basaldiet-fed controls (FIG. 3B). Cyclin-D1 staining in hepatocytes wasincreased in both groups as compared to basal diet (FIG. 3B). However,when BrdU-positive hepatocytes were counted, a significant difference inBrdU incorporation was continuously observed between T3 versus GC-1 dietgroups (FIG. 3C), although the discrepancy was less than what wasobserved in T3 diet fed versus GC-1 injected group. Thus, GC-1 induceshepatocyte proliferation via β-catenin activation and cyclin-D1expression, although to modestly less extent than T3.

Example 4 Presence of S-Catenin is Required for Significant Level ofProliferation in Response to TRβ Agonist GC-1

T3 hormone has been shown to require β-catenin to exert mitogenic effectin mouse hepatocytes (Fanti et al. (2014) Hepatology 59, 2309-2320).Thus, it was determined whether the action of selective TRβ agonistwould similarly be dependent on β-catenin. β-Cat LKO mice and wild typelittermate controls received eight daily IP injections of GC-1 (0.3mg/Kg/dose) or DMSO alone (control) as described in methods. Similar toT3, GC-1 treated β-Cat LKO mice showed decreased hepatocyteproliferation as shown by low BrdU incorporation in hepatocytes in β-CatLKO as compared to controls (FIGS. 4A-B). However, notable BrdUincorporation continued to occur in the non-parenchymal cell populationin the β-Cat LKO group after GC-1 administration. Both findings weresimilar to those observed after T3 administration to β-Cat LKO (Fanti etal., supra).

Consistent with the lack of optimum BrdU response to GC-1, the β-Cat LKOlivers also showed a notable decrease in nuclear cyclin-D1 byimmunohistochemistry when compared to controls, similar to that observedin response to T3 (FIG. 4C). This was verified by RT-PCR analysis, whichshowed a significant decrease in cyclin-D1 mRNA expression inβ-catenin-LKO mice receiving GC-1 compared to controls (FIG. 4D).

Lastly, the status of PKA-dependent serine phosphorylation of β-cateninin β-Cat LKO was addressed following GC-1 and T3 treatment. Western blotanalysis using liver lysates from β-Cat LKO and control mice treatedwith T3 or GC-1 showed notably lower total β-catenin in KO, whichrepresents continued β-catenin expression in the non-parenchymal cellsas also noted elsewhere (Tan et al. (2006) Gastroenterology 131,1561-1572) (FIG. 4E). As expected, P-Ser675-β-catenin was absent inβ-Cat LKO as compared to controls treated with GC-1 or T3. Intriguinglyit was found that β-cat LKO mice that received T3 or GC-1 showed evenhigher levels of P-Ser552-β-catenin levels as compared to controlssubjected to similar treatment (FIG. 4E). These findings suggest adivergence in PKA-dependent β-catenin activation in response to T3 orGC-1 in hepatocytes versus non-parenchymal cells that may eventually becontributing to their overall mitogenic response in the liver.

Example 5 Lack of Hepatocyte Proliferation in Response to Both T3 andGC-1 Upon Disruption of Wnt-β-Catenin Axis in the Hepatocytes

Previous work and the work disclosed herein show that like T3, GC-1 alsoincreases phosphorylation of β-catenin at Ser675. To furthersubstantiate these results, it was directly investigated ifWnt-dependent β-catenin activation could be contributing to the overallmitogenic effect of T3 and GC-1. For this, LRP5-6 LKO mice were utilizedthat lack the two redundant Wnt co-receptors (LRP5 and LRP6). Thesemice, unlike β-Cat LKO have intact β-catenin in hepatocytes, however theabsence of Wnt co-receptors, disallows β-catenin activation inhepatocytes in response to Wnt secretion (Fanti et al., supra).

LRP5-6 LKO male mice and littermate controls aged 2-3 months receivedeither T3 supplemented diet (4 mg/Kg of diet) or regular control diet.Another cohort received 8 daily IP injections of GC-1 (0.3 mg/Kg/dose)or DMSO. Interestingly, LRP5-6 LKO mice treated with either T3 or GC-1showed a notable decrease in the number of BrdU-labeled hepatocytenuclei when compared with the controls (FIG. 5A). A careful counting ofthe BrdU-positive hepatocytes verified a significant difference inhepatocyte proliferation in response to T3 and GC-1 as compared tocontrols (FIG. 5B), and occurred with the same profoundness in responseto T3 or GC-1 in the absence of LRP5-6 in hepatocytes (FIG. 5C).Likewise, the numbers of cyclin-D1-positive hepatocytes was also notablydecreased in LRP5-6 LKO in response to T3 and GC-1 as shown byimmunohistochemistry (FIG. 5A).

The mechanism of this unexpected finding was explored. It was determinedif β-catenin levels were any different in LRP5-6 LKO. As expected,comparable levels of total β-catenin was found in the LRP5-6 LKO incontrol and T3 group (FIG. 6A) and GC-1 group. Further, in agreementwith immunohistochemistry findings (FIG. 5A), LRP5-6 LKO showeddecreased cyclin-D1 protein and mRNA expression in response to both T3and GC-1 by Western blots as well (FIGS. 6A, 6B, and 6C). Interestingly,either comparable or even greater levels of pSer675-β-catenin wereevident in LRP5-6-LKO and littermate controls treated with GC-1 and T3(FIGS. 6A, 6B). There was a modest increase in the levels ofpSer552-β-catenin in LRP5-6 LKO after both T3 and GC-1 (FIGS. 6A, 6B).

Because cyclin-D1 expression is regulated by β-catenin interaction withtranscription factor TCF4, the liver lysates were assayed from theLRP5-6 LKO for the levels of TCF4. Comparable levels of total TCF4 wereevident in the LRP-LKO and controls (FIG. 6D).

Because β-catenin-LKO and LRP5-6 LKO both are refractory to T3 and GC-1treatment in terms of hepatocyte proliferation due to impaired cyclin-D1induction, it was determined whether intact Wnt signaling was alsocontributing to β-catenin activation in addition to PKA-dependentβ-catenin activation, as evidenced by increased pSer675-O-catenin. Forthis study, an antibody against active β-catenin (hypophosphorylated atSer33, Ser37 and Thr41) was used. To check the specificity of thisantibody, liver lysates were used from β-Cat LKO and controls treatedwith T3, GC1 or basal diet. A notable increase in active-β-catenin wasevident in GC-1 treated group as compared to basal diet (FIG. 6E). Avery faint signal presumably due to non-parenchymal cells, was evidentin J-Cat LKO after T3 or GC-1 treatment indicating the specificity ofthe antibody (FIG. 6E). Next, it was determined if intact Wnt signalingis required to activate β-catenin in hepatocytes after T3 or GC-1treatment. A dramatic increase in active β-catenin levels was evident inliver lysates of control mice fed T3 or GC-1 diet for 8 days as comparedto basal diet fed mice (FIG. 6F). However, no notable differences inactive β-catenin levels were evident in LRP5-6 LKO after T3 or GC-1feeding when compared to basal diet (FIG. 6F).

Taken together, these findings suggest that hepatocyte proliferation inresponse to both T3 and GC-1 may eventually be a net result of PKA- andWnt-dependent $-catenin activation.

Example 6 Pretreatment with T3 or GC-1 Supplemented Diet Prior toPartial Hepatectomy Leads to a Proliferative Advantage at 24 Hours PostHepatectomy in Mice

To directly investigate any significance of T3 or GC-1 in hepaticregenerative therapies, their usage in partial hepatectomy model wasinvestigated. Because the kinetics of hepatocyte proliferation duringliver regeneration after partial hepatectomy in C57BL/6 mice are wellknown, it was determined if T3 or GC-1 pre-treatment of mice offers anyregenerative advantage in the setting of partial hepatectomy, aprocedure relevant in patients as well. Two-month old C57BL/6 mice wereput on T3-diet, GC-1-diet or kept on basal diet for 7 days with drinkingwater containing BrdU ad libitum. On day 7, all mice were subjected totwo-thirds hepatectomy and resected lobes retained for analysis for BrdUand cyclin-D1 by immunohistochemistry. Hepatectomized mice from allthree groups were sacrificed 24 hours later, a time-point around 12-16hours prior to the observed peak hepatocyte proliferation (Fausto (2000)J Hepatol 32, 19-31; Michalopoulos (2013) Compr Physiol 3, 485-513).Livers from 24 hour time point were assessed for BrdU incorporation inhepatocytes and cyclin-D1 by immunohistochemistry. Seven-daypretreatment of mice with either T3 or GC-1 led to increasedBrdU-positive hepatocytes as compared to basal diet as observed in theanalysis of resected lobes (FIG. 7A). Interestingly, while very fewhepatocytes showed BrdU positive nuclei at 24 hours after hepatectomy inmice fed basal diet, a pronounced increase was observed in T3 as well asGC-1-fed group of animals (FIG. 7A). In fact, the numbers of hepatocytespositive for nuclear BrdU were significantly higher in T3 and GC-1 fedmice versus basal diet (FIG. 7B). T3 pretreatment showed significantlygreater hepatocyte proliferative response than GC-1 post hepatectomy(FIG. 7B).

Cyclin-D1 was assessed in the same groups by immunohistochemistry.Cyclin-D1 was restricted to a subset of midzonal hepatocytes in basaldiet fed mice both in resected lobes as well as regenerating lobes at 24hours (FIG. 7C). An expansion of cyclin-D1 positive hepatocytes in themidzonal area was evident in both T3 and GC-1 pretreatment livers in theresected lobes at the time of hepatectomy (FIG. 7C). However, at 24hours after hepatectomy, a panlobular distribution of hepatocytes withnuclear cyclin-D1 was clearly evident in the livers from T3 as well asGC-1 treated mice. (FIG. 7C).

Finally, change in BrdU incorporation was determined longitudinally inthe same animals in basal diet, T3 and GC-1 groups to address theirrelative efficacy in inducing hepatocyte proliferation over 24 hoursduring liver regeneration. No significant differences were observed inhepatocyte proliferation between pre-hepatectomy and 24 hour-posthepatectomy livers in animals that were on basal diet (FIG. 8A). T3induced significant increase in hepatocyte proliferation which wasaround 2-fold over the 24-hour liver regeneration period in all threemice tested (FIG. 8B). Similarly, GC-1 also induced a significantincrease in hepatocyte proliferation of around 2-fold over its baselinewithin 24 hours during liver regeneration in all three animals tested(FIG. 8C). Thus both T3 and GC-1 pretreatment offered a regenerativeadvantage to the liver following surgical resection in mice.

Thus, selective thyromimetics activating TRβ in the liver are effectivein inducing hepatocyte proliferation. The hepatocyte proliferationinduced by GC-1 could be dependent on the presence of β-catenin. Indeed,the results show that GC-1 is capable of inducing β-catenin activation,which in turn increased Cyclin-D1 expression and eventually hepatocyteproliferation, when given either as an injection or as a diet.Intriguingly, GC-1 when delivered IP in DMSO showed lesser increases inhepatocyte proliferation than its delivery orally through diet. This wasmost likely due to the use of DMSO as a solvent for GC-1. This wasstrengthened by the observation that DMSO delivery by itself affectedhepatocyte proliferation. Indeed, the role of DMSO in inhibitinghepatocyte proliferation especially in cultures has been known for along time (Chan et al. (1989) J Cell Physiol 141, 584-590). When GC-1was administered to mice via diet, hepatocyte proliferation wasincreased to a notably greater extent, although it was less so than thatinduced with T3-diet. This could be due to the dose of GC-1 in diet orother mechanisms, especially since a previous study has reportedcomparable efficacy of T3 and GC-1 in inducing hepatocyte proliferation(Kowalik et al. (2010) J Hepatol 53, 686-692). Nonetheless, GC-1administration caused a profound increase in hepatocyte proliferation.

This study also elucidated the mechanism by which T3 and selectivethyromimetics cause hepatocyte proliferation. In order to identify ifβ-catenin is also required for the actions of GC-1, GC-1 wasadministered to β-cat-LKO mice and controls. The results showed that,similar to T3, there was a significant decrease in proliferation inresponse to GC-1 when β-catenin is absent from hepatocytes. Thisoccurred concomitant to lack of Cyclin-D1 increase in hepatocytes, whichis a known β-catenin target (Tan et al., supra). To further try andcharacterize the mechanism of β-catenin activation by the thyromimeticslike T3 and GC-1, a mouse model was used that has conditional loss ofWnt co-receptors LRP5 and LRP6 in hepatocytes and has been described byus recently (Yang et al. (2014) Hepatology 60, 964-976). In the absenceof these co-receptors, Wnt is unable to transduce its signal toβ-catenin (Riddle et al. (2013) PloS one 8, e63323). However, β-cateninis present in hepatocytes and hence may be capable of being activated bynon-Wnt mechanisms. Based on the previously proposed mechanism involvingPKA-dependent phosphorylation of β-catenin, treatment of LRP5-6 LKO micewith T3 or GC-1 could result in a similar degree of hepatocyteproliferation when compared to the control mice. Intriguingly, theresults show that LRP5-6 LKO mice have significantly reduced hepatocyteproliferation in response to T3 or GC-1 when compared to controls alongwith decreased Cyclin-D1 in hepatocyte nuclei. Furthermore, thisoccurred despite comparable levels of TCF4 and equally increased levelsof pSer675-β-catenin in LRP5-6 LKO mice. This suggests that T3 and GC-1induced β-catenin activation brought about via PKA activation is intactin the absence of LRP5-6 in hepatocytes. To address the discrepancy oflow regenerative response in LRP5-6 LKO following GC-1 and T3, theeffect of these factors on canonical Wnt signaling was revisited. Theeffect of T3 on Wnt-dependent β-catenin activation was previouslystudied by only assessing any changes in levels of Ser9-GSK30 (Fanti etal., supra). However, this phosphorylation site at GSK30 has been shownto be inconsequential in Wnt signaling and only relevant in Insulinsignaling (McManus et al. (2005) The EMBO journal 24, 1571-1583). In thestudies disclosed herein an antibody was used that detects β-cateninwhich is hypophosphorylated at Ser33, Ser37 and Thr41, which is a directconsequence of Wnt signaling (Villar et al. (2011) PloS one 6, e23914).The specificity of this antibody was verified using β-catenin LKO.Further, a notable increase in active-β-catenin levels following GC-1treatment in normal animals was observed. In absence of LRP5-6 onhepatocytes, there was a failure of any increase in active-β-cateninlevels in response to T3 and GC-1. Without being bound by theory, thesefindings suggest that T3 and TRβ-agonists require both intact Wntsignaling as well as PKA to allow for optimal β-catenin activation andhepatocyte proliferation (FIG. 9).

The mechanism whereinT3 or GC-1 cause Wnt-dependent β-catenin activationwas investigated. In liver regeneration after partial hepatectomy,non-parenchymal cells (NPC) such as sinusoidal endothelial cells andmacrophages are the source of Wnts which activate β-catenin inhepatocytes in a paracrine manner (Yang et al. (2014) Hepatology60,964-976; Ding et al. (2010) Nature 468, 310-315). NPC proliferationin response to T3 and GC-1 was observed. Thus, thyromimetics may also becontributing to β-catenin activation and hepatocyte proliferationindirectly by inducing Wnt secretion from NPCs (FIG. 9).

An increase in the levels of pSer552-β-catenin was observed inβ-catenin-LKO and LRP5-6-LKO mice following treatment with T3 and GC-1,which was unexpected. This site can also lead to β-catenin activation(Taurin et al., (2006) J Biol Chem 281, 9971-9976). Intriguingly, thisincrease was evident even in livers that lacked β-catenin in hepatocytesand hence this observation most likely represents β-cateninphosphorylation and activation in the non-parenchymal cells of the liverfollowing GC-1 and T3 treatment. Without being bound by theory, it isconceivable that T3 and GC-1 may be stimulating proliferation ofnon-parenchymal cells such as endothelial cells by β-catenin activationthrough phosphorylation at Ser552. Also, thyromimetics may inducerelease of Wnt proteins from endothelial cells, which could themcontribute to eventually contribute to endothelial cell proliferation inan autocrine manner and to hepatocyte proliferation in a paracrinefashion through stimulation of the canonical Wnt signaling pathway (FIG.9).

The last part of the disclosed study directly investigated whetherpretreatment with T3 or GC-1 prior to partial hepatectomy would confer aregenerative advantage to mice. The effect of T3 on liver regenerationhas been examined in rats previously, and those results did show aregenerative advantage after partial hepatectomy, and a survivaladvantage in a 90% hepatectomy model, which is normally associated withhigh mortality (Columbano et al. (2008) Cell Prolif 41, 521-53). Theresults show significantly increased BrdU incorporation in hepatocytes24 hours after partial hepatectomy in mice that received T3- orGC-1-supplemented diet for 8 days prior to the surgery. Even though thisis a nonlethal model and mouse livers typically regenerate within 14days, a clear and robust regenerative response was shown following T3 orGC-1 pre-administration. This was again due to increased expression ofCyclin-D1. This part of the study demonstrates the direct relevance ofadministration of thyromimetics in the setting of liver transplantationto induce regeneration in the donor and/or recipient.

Thus, like T3, GC-1 causes pronounced hepatocyte proliferation secondaryto increased Cyclin-D1 expression that is dependent on activation ofβ-catenin. It was found that disrupting Wnt signaling abolishes GC-1-and T3-dependent β-catenin activation. The efficacy of these agents toinduce hepatocyte proliferation and stimulate the process of liverregeneration was validated, which has significant therapeuticimplications in the transplantation settings.

Example 7

The mitogenic effects of TRβ-selective agonist GC-1 in hepatocyteregeneration and proliferation in liver donors are determined prior tohepatectomy. The effectiveness of GC-1 therapy, when given prior tohepatectomy in the selected liver donors leads to acceleratedphysiological regeneration, and provides a proliferative advantage whencompared to the control group. GC-1 is also used in patients post livingdonor recipients. GC-1 accelerates the liver regeneration orproliferation. GC-1 is also used in post liver transplant patients withSmall for Size syndrome. In addition, GC-1 is used in liver transplantrecipients who received extended criteria livers.

Example 8

The effects of GC-1 are evaluated on lipid profile and liver functiontests post hepatectomy. Ancillary studies are performed of thepathogenesis, diagnosis or diagnostic biomarker development, naturalhistory and treatment of GC-1. In addition, the following isinvestigated:

1. Non-alcoholic fatty liver disease (NAFLD)/or liver injury for otherreasons

2. Acute Alcoholic Hepatitis (AAH)

3. Fulminant Hepatic Failure (FHF)

4. Tylenol induced liver injury (LI) or

5. Patients post liver transplant (LT) with cholestasis or Drug InducedLiver Injury (DILI) or Total Parenteral Nutrition (TPN) induced liverinjury (LI).

Example 9

An intravenous (IV) infusion is used, such as GC-1 (0.3 mg/kg/dose)dissolved in a solvent (DMSO). GC-1 is administered Day #1 afterhepatectomy in donors or hepatectomy for other reasons. The duration canbe one, two or three weeks. In some examples, subject who are livertransplant recipients from extended criteria donors are treated.

A liver biopsy is taken during the initial procedure. Imaging andlaboratory tests are used to determine synthetic/metabolic functionpost-operatively in the non-GC-1 administered control group Vs GC-1group and follow the liver proliferation and regeneration.

Example 10 Partial Hepatectomy Study in Mice

Seven days prior to surgery, mice are initiated on GC-1 containing diet(5 mg/kg). Diet is made available ad libitum. Surgery is performed atday 7. The mice continue to be on GC-1 diet. Three mice will besacrificed at 12 hours, 3 mice at 24 hours, 3 mice at 48 hours, 3 miceat 7 days and 3 mice at 14 days. Blood is analyzed at all these timesfor serum albumin, serum ALT levels, serum AST levels, prothrombintime/INR (international normalized ratio), bilirubin and glucose andlipid profiles. Livers harvested at each time point are assessed forweight (body weight), hepatocyte proliferation, Wnt target genes andgene expression studies. These studies directly test prolonged benefitof GC-1 on liver regeneration.

Example 11 Promoting Regeneration to Treat Hepatic Steatosis

Long term regeneration in mice is associated with steatosis (one yearafter partial hepatectomy). GC-1 is used for 10 days after partialhepatectomy only and then mice are followed for 1 year to examinesteatosis. In second group of mice, surgery is performed and 11.5 monthsafter surgery, and these mice are randomized into 2 groups—one is put onGC-1 diet and other on regular diet for 15 days before sacrificing. Thelivers are examined for the same parameters as in Example 11 and areexamined for fat deposition by H&E as well as oil red o staining. Inaddition, serum and liver are examined for triglycerides, HDL, VLDL andcholesterol. Increased hepatocyte proliferation, improved serumbiochemistry and decreased serum and hepatic lipids in GC-1 group showan advantage of use of GC-1 in steatosis by promoting regeneration.

Example 12 Acetaminophen (APAP) Overdose

a. Sublethal dose: Mice are on GC-1 diet or basal diet for 7 days. Onday 7, they are overnight fasted and injected intraperitoneally with asublethal dose of APAP at 300 mg/kg body. Mice (n=3/timepoint/group-basal diet versus GC-1) are sacrificed at 6 hours (h) and 12h. Additional mice from each group are provided GC-1 or basal diet after6 hours of APAP injection and 3 mice from each group are killed at 24nand 48 h to test for serum albumin, serum ALT levels, serum AST levels,prothrombin time/INR (international normalized ratio), bilirubin andglucose and lipid profiles. Liver weight/body weight ratio is assessedand liver histology and markers for liver regeneration are assessed atall times. Increased hepatocyte proliferation, improved liver weightsand improved serum biochemistry in GC-1 group advantage of use of GC-1in APAP overdose.

b. Lethal dose: Mice are on GC-1 diet or basal diet for 7 days. On day7, they are overnight fasted and injected intraperitoneally with alethal dose of APAP at 450 mg/kg body. Mice (n=3/time point/group-basaldiet versus GC-1) are sacrificed at 6 h and 12 h. Additional mice fromeach group are provided GC-1 or basal diet after 6 hours of APAPinjection and 3 mice from each group are killed at 24 h and 48 h to testfor serum albumin, serum ALT levels, serum AST levels, prothrombintime/INR (international normalized ratio), bilirubin and glucose andlipid profiles. Liver weight/body weight ratio is assessed and liverhistology and markers for liver regeneration are assessed at all times.Mice are expected to perish in the basal diet group by 12-24 h. Survivaladvantage will be assessed by Kaplan Meier analysis of GC-1 versus basaldiet groups. Increased hepatocyte proliferation, improved liver weights,improved survival and improved serum biochemistry in GC-1 groupadvantage of use of GC-1 in APAP overdose.

Example 13 Clinical Trial Role for the Potential Applications inTransplant Population:

There is an unmet clinic need and utility in transplant population inareas of liver regeneration and liver regeneration. GC-1 can be utilizedin a transplant population including:

-   -   Living donors prior to donor surgery    -   Living donor recipients    -   Post hepatectomy patients for other reasons like        Hepatoblasotoma, hepatic adenoma etc.    -   Post liver transplant cadaveric recipients with SFSS (Small for        size syndrome)    -   Extended criteria donor livers including DCD donors given the        scarcity of organs and increased wait list mortality    -   Utility in high risk donors: Marginal or extended criteria        donors (ECD) are defined as those with a greater risk of initial        poor function or graft failure and therefore an increased risk        for recipient morbidity and mortality    -   The features of a marginal organ have not been clearly defined,        although some circumstances are known to be related to impaired        graft function: elderly donors, a high grade of steatosis,        DCD/non-heart-beating donors, or split grafts    -   In donors with high DRI (Donor risk index)

Dosage of GC-1

-   -   Intravenous (IV) infusion    -   GC-1 (0.3 mg/kg/dose) dissolved in a solvent (DMSO or saline) as        an IV infusion, typically on Day #1 after hepatectomy in donors        or hepatectomy for other reasons    -   Patients who are liver transplant recipients from extended        criteria donors    -   Duration: 2 weeks    -   Liver biopsy intra op and imaging and other labs to check        synthetic/metabolic function post operatively.    -   Control Vs GC-1 group and follow the liver proliferation and        regeneration

Study Overview

Compare liver regeneration in 10 patients who will be IV infused GC-1(0.3 mg/kg/day)×7 days starting day 1 after transplantation to 10patients without GC-1. Liver regeneration will be measured byradiological monitoring of hepatic size and hepatic function (serumalbumin, Prothrombin time/INR (international normalized ratio),bilirubin, ALT and AST levels).

Example 14 Materials and Methods for Examples 15-21

Because GC-1 is of relevance as regenerative therapy both intransplantation settings, it is pertinent to directly address its effecton tumor growth and development, especially those that are driven by theWnt/β-catenin signaling pathway. The effect of GC-1 on liver tumorcells, and in a HCC model driven by the co-expression of S45Y-β-cateninand hMet using SBTT and HTVI (Tao et al., Hepatology 2016, 64:1587-605),was assessed. It was demonstrated that GC-1 decreased tumor burden owingto decreased tumor cell proliferation with a notable decrease in Met-Erkand Met-Stat3 signaling and no effect on Akt or Wnt/β-catenin signaling.Thus, GC-1 suppresses tumorigenesis and does not enhance Wnt/β-cateninin HCC, demonstrating its overall safety for use in chronic liverdiseases and after transplantation, to induce regeneration.

The following materials and methods were used in these studies:

Animals, Plasmids and Hydrodynamic tail vein injections. SBTT plasmidsand HTVI have been described previously (Tao et al., Hepatology 2016,64:1587-605). Briefly, 20 μg pT3-EF5α-hMet-V5 andpT3-EF5α-S45Y-β-catenin-Myc combination along with the transposase in aratio of 25:1 were diluted in 2 ml of normal saline (0.9% NaCl),filtered through 0.22 μm filter (Millipore), and injected into thelateral tail vein of 23 FVB mice that were around 6-week-old, in 5-7seconds. These mice are referred henceforth as hMet-mutant-β-cateninmice. Four weeks after injection, hMet-mutant-O-catenin mice wererandomized into two groups. One group was kept on basal diet (n=12) andanother group was switched to GC-1-supplemented diet (5 mg/kg of diet,Medchem Express) (n=11). Animals on control diet were sacrificed ateither 21 days (n=8) or 10 days (n=4) after initiation of diet.Similarly, animals on GC-1-diet were sacrificed at either 21 days (n=7)or 10 days (n=4) after initiation of the diet. The animals were givenaccess to food and water ad libitum with a 12-hour light/dark dailycycle. One intraperitoneal injection of BrdU was performed on day 9during 10 days of GC-1 or basal diet treatment and livers were harvested24 hours later. Guidelines for the Care and Use of Laboratory Animalswere followed.

Immunohistochemistry. Four micron formalin-fixed sections weredeparaffinized in graded xylene and alcohol and rinsed in PBS. To blockendogenous peroxidase activity, the sections were incubated in 3%hydrogen peroxide (Sigma). For antigen retrieval, slides were microwavedin citrate buffer followed by blocking with Superblock (ScyTekLaboratories, Logan, Utah) for 10 minutes. Sections were incubatedovernight at 4° C. or 1 hour room temperature in the followingantibodies: cyclin-D1 (Thermo-Scientific, Fremont, Calif.), Glutaminesynthetase, Ki-67, Myc-tag and CD45 (Santa Cruz Biotechnology, Dallas,Tex.). Sections were then incubated with species-specific secondaryhorseradish peroxidase-conjugated antibody for 30 minutes at roomtemperature. Sections stained with antibodies were incubated withstreptavidin-biotin and signal was detected with DAB. Cell death wasevaluated in liver sections by terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL) using manufacturer's instructionsavailable with the kit (EMD-Millipore). BrdU was stained with mouseantibody (Becton Dickinson, Franklin Lakes, N.J.) as previouslydescribed (Alvarado et al., Gene Expr 2016, 17:19-34). Briefly, tissuesections were deparaffinized, exposed to 0.3% hydrogen peroxide indeionized water for 10 minutes to block endogenous peroxidase, treatedwith 2N HCl, incubated with trypsin 0.1% for 20 minutes and then withnormal goat serum for 20 minutes at room temperature. Sections were thenincubated overnight in cold room with anti-BrdU monoclonal antibody,followed by biotinylated goat anti-mouse IgG. DAB kit was then applied,and sections were counterstained with hematoxylin.

For quantification of Ki-67 immunohistochemistry (IHC), pictures weretaken at 200× magnification from either tumor or non-tumor areas in thesame slide. Each picture was separated into DAB staining channel andhematoxylin staining channel by Color Deconvolution using Imagej Fiji.To quantify the number of Ki-67 positive nuclei on each picture, the DABstaining on the DAB channel was highlighted with the threshold of 48 andthen quantified with Analyze Particles. All particles of areas smallerthan 100 pixels were excluded. To quantify the total number of nuclei oneach picture the hematoxylin staining on the hematoxylin channel washighlighted with the threshold of 205 and then quantified with AnalyzeParticles with similar exclusion of small areas. The percentage ofKi-67-positive nuclei on each picture was calculated by dividing thenumber of particles counted from DAB channel by the number of particlescounted from the hematoxylin channel.

Protein Extraction and Western Blot Analysis. Flash-frozen livers fromhMet-mutant-β-catenin FVB mice on control/basal diet or GC-1-diet wereused to obtain whole cell lysates. For protein extraction, a smallamount of liver tissue was homogenized using RIPA buffer containing theprotease and phosphatase inhibitor cocktail (Sigma, St. Louis, Mo.).Tissue homogenate was centrifuged at 14,000 RPM for 5 minutes in coldroom. Supernatant was recovered and stored at −80° C. for use. Aliquotsof 30-50 μg of proteins were denatured by boiling in Tris-Glycine SDSSample Buffer (Life Technologies, Carlsbad, Calif.), resolved by SDSPAGE, and transferred to PVDF membranes (Life Technologies) using theBiorad transfer apparatus. Membranes were blocked in 5% non-fat dry milkor 5% BSA in Tris-buffered saline containing 0.1% Tween 20 for 1 hour.Western blot analysis was performed using the following primaryantibodies Active-β-catenin (1:800, Cell Signaling, Danvers, Mass.),cyclin-D1 (1:1000, Thermo-Scientific), GS (1:2000, Santa CruzBiotechnology), ERK1/2 (1:1000, Cell Signaling), P-ERK1/2 (T202, Y204)(1:1000, Cell Signaling), P-MET (Y1234/1235) (1; 1:500, Cell SignalingCST 3077S), P-MET (Y1234/1235) (2; 1:500, Cell Signaling CST 3129S),Total MET (1:500, Cell Signaling), STAT3 (1:500, Santa Cruz), P-STAT3(Y705) (1:100, Cell Signaling), AKT (1:1000, Cell Signaling) and P-AKT(S475) (1:1000, Cell Signaling). Membranes were incubated in primaryantibodies, diluted in 5% skim milk or 5% BSA following overnightincubation at 4° C. Incubation with anti-rabbit or anti-mouse secondaryantibody horseradish peroxidase-conjugated immunoglobulin G (IgG; SantaCruz Biotechnology, Santa Cruz, Calif., USA) was done for 30 minutes atroom temperature. Immunoreactive bands were detected by Super-SignalWest Pico Chemiluminescense Substrate (ThermoFisher Scientific, RockfordIll.) and revealed by autoradiography.

Real Time Polymerase Chain Reaction (RT-PCR). Isolation of total RNA wasperformed using Trizol reagent (Invitrogen, Carlsbad, Calif.), fromfrozen liver tissue. Aliquots containing 2 μg of total RNA werereverse-transcribed after DNAse enzymatic treatment to remove genomicDNA contamination, using Super Script III first strand kit (ThermoFisherScientific). Real-time PCR was performed on an ABI Prism 7300 SequenceDetection System (Applied Biosystems, Foster City, Calif.) usingSybrgreen. Deiodinase I values were normalized to GAPDH.

TopFlash Reporter Assay. Three liver tumor cell lines Hep3B, HepG2 andSnu-398 cells were obtained from ATCC (Manassas, Va.). Cells weretransfected simultaneously with Renilla reniformis luciferase (pRL-TK;Promega, Madison, Wis.) as a transfection control and TopFlash fireflyluciferase plasmids (Upstate Biotechnology, Lake Placid, N.Y.), whichcontains three copies of the Tcf/Lef sites upstream of a thymidinekinase (TK) promoter and the firefly luciferase gene using Lipofectamine2000 (Life Technologies). Twenty-four hours after transfection, cellswere treated with either DMSO (Fisher Scientific) or 5-7 μM GC-1(Medchem Express, Monmouth Junction, N.J.) for 24 hours. Lysates wereharvested using the Dual-Luciferase Reporter Assay System (Promega,Madison, Wis.). Firefly luciferase signals were normalized to Renillaluciferase and ratio between groups compared by student's t-test todetermine significance. P<0.05 was considered significant.

Statistical analysis. All statistics were performed using the Prism 6for Mac OS X software (Version 6) (GraphPad Software, Inc.) and thecomparison between treated and control group was performed by Student'st test. P<0.05 was considered significant (*), p<0.01 was consideredhighly significant (**) and p<0.001 was considered extremely significant(***).

Example 15 GC-1 Treatment does not Enhance β-Catenin-TCF4 ReporterActivity in CTNNB1-Mutated and Non-Mutated Human HCC Cells

GC-1 has been found to stimulate hepatocyte proliferation at least inpart through the activation of Wnt/β-catenin signaling (Alvarado et al.,Gene Expr 2016, 17:19-34; Fanti et al., Hepatology 2014, 59:2309-20).Since Wnt/β-catenin activation due to mutations in key effectors of thepathway is reported in a significant subset of HCC cases, it wasaddressed if GC-1 could promote β-catenin-TCF4 activity in various livertumor cells. The effect of GC-1 treatment was directly examined on threedifferent liver tumor cell lines which normally contain wild-type CTNNB1(Hep3B cells), point-mutant β-catenin (Snu-398 cells) andexon-3-deletion mutant of CTNNB1 (HepG2 cells). TopFlashreporter-transfected cells were treated for GC-1 as indicated inmethods. GC-1 treatment had no significant effect on the TopFlashluciferase reporter activity in any of the three cell lines as comparedto the respective DMSO-treated controls (FIGS. 10 A-10F). Thus,irrespective of the status of β-catenin gene mutations, GC-1 does notincrease or decrease Wnt/β-catenin activity in various HCC cell lines.

Example 16 Three-Week Treatment with GC-1 Decreases Tumor Burden inhMet-S45Y-β-Catenin HCC Model by Decreasing Tumor Cell Proliferation

To further address any effect of GC-1 on HCC in vivo in a model whichrepresents a clinical disease, and is driven by combination of twoproto-oncogenes-mutant-CTNNB1 and hMet, a recently described murinemodel (Tao et all, Hepatology 2016, 64:1587-605) was employed.hMet-S45Y-β-catenin mice were randomized into two groups, one receivedbasal and another GC-1-diet for 21 days (FIG. 11A). The effectiveness ofGC-1 was verified by examining hepatic expression of deiodinase 1 (Diol)gene, a surrogate target of THRβ (Bianco et al., J Clin Invest 2006,116:2571-9; Gullbeg et al., Mol Endocrinol 2002, 16:1767-77), which wassignificantly upregulated in GC-1-treated group by RT-PCR (FIG. 11B).GC-1's effect on overall tumor burden was next assessed by comparing theliver weight to body weight ratios (LW/BW×100; percent) in the twogroups. An almost significant decrease (p=0.050⁶) in LW/BW was evidentin the GC-1 group (FIG. 11C). Grossly, most livers from basal diet groupshowed large tumors and irregular surface depicting notabletumorigenesis, while all livers from GC-1 treated groups showedrelatively smooth surface and smaller nodules (FIG. 11D). H&E stainingof livers from basal diet-fed mice after 7 weeks of HTVI showed severalabutting tumor foci with only a few layers of normal hepatocytescompressed in between (FIG. 12A). This was clearly evident inimmunohistochemistry (IHC) for Myc-tag in these liver sections (FIG.12B), as also shown previously (Tao et al., Hepatology 2016,64:1587-605). The GC-1 diet-fed hMet-S45Y-β-catenin mice showed notablysmaller tumor nodules interspersed among normal hepatocytes as seen byboth H&E and Myc-tag IHC (FIGS. 12A-12B). A modest decrease in Myc-taglevels by Western blot (WB) analysis also verified overall lower tumorburden in these group of animals (FIG. 12C).

To address the basis of smaller tumor foci in hMet-S45Y-β-catenin miceafter GC-1 versus control diet, a terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL) was performed as a marker of cell death.More TUNEL-positive cells were evident in the basal diet group ascompared to GC-1 group likely due to excessive tumor size, precludingincreased cell death to be mechanism of lower tumor burden (FIG. 12D).

Next, IHC for Ki-67, marker of cells in S-phase of cell cycle, wasperformed. While tumor nodules were smaller in the liver sections of theGC-1 treated group, several cells were positive for Ki-67 stainingwithin the tumor nodules as compared to basal diet, where relativelyfewer tumor cells within large nodules were positive (FIG. 13A).Quantification of Ki-67 IHC revealed insignificant differences in thepercentage of positive tumor cells within nodules between the two groups(FIG. 13B). Likewise, non-tumor areas of both group showed scantKI-67-positive cells and differences between GC-1 and basal diet groupwere insignificant (FIG. 13B).

Example 17 Three-Week Treatment with GC-1 in hMet-S45Y-β-Catenin HCCModel does not Impact Wnt Signaling

To address the molecular basis of reduced tumor burden, it was firstassessed if GC-1 could have any paradoxical and negative effect on Wntsignaling pathway since it is known to promote Wnt signaling in thecontext of cell proliferation and liver regeneration (Alvarado et al.,Gene Expr 2016, 17:19-34; Fanti et al., Hepatology 2014, 59:2309-20).The status of tumor nodules was assessed in each group for Wnt/β-cateninpathway targets such as cyclin-D1 and Glutamine synthetase (GS) by IHC.Tumor nodules in basal diet group were strongly positive for cyclin-D1as were the nodules in GC-1 diet, despite being smaller in the lattergroup (FIG. 14A). Cyclin-D1 was localized to both tumors as well assurrounding normal tissue in both groups. The tumors were uniformly andcomparably positive for GS by IHC in both groups despite the smallersize of nodules in the GC-1 group (FIG. 14B). GS was localized mostly inthe tumors in both groups.

To validate IHC findings, Western Blot (WB) analysis was performed onlysates from tumor-bearing livers from both groups for Wnt signalingcomponents. Total and active β-catenin levels remained unaltered in thetwo groups (FIG. 14C). Similarly, no change in cyclin-D1 levels wereevident by WB (FIG. 14C). A modest decrease in GS levels by WB in theGC-1 group may also represent a decrease in tumor burden since likeMyc-tag, GS was predominantly expressed in tumor nodules only (FIG.14C). Thus, GC-1 had no impact on Wnt/β-catenin signaling inhMet-S45Y-β-catenin mice and did not increase this signaling pathway orpromote hepatocellular carcinoma (HCC) burden.

Example 18 Three-Week Treatment with GC-1 in hMet-S45Y-β-Catenin HCCModel Impairs Met-Erk and Met-Stat3 Signaling

Because HCC in the hMet-S45Y-β-catenin mice is due to functional andsynergistic cooperation of the two proto-oncogenes (Tao et al.,Hepatology 2016, 64:1587-605) and because GC-1 did not alter Wntsignaling, its effect was next tested on Met signaling. A dramaticdecrease in p-Met (Tyr 1234/1235) was observed in GC-1-treated group ascompared to the basal diet controls (FIG. 15A). Total Met levels wereonly marginally and variably decreased after GC-1 treatment (FIG. 15A).

Since Met signaling was recently shown to predominantly act throughdownstream Ras-Erk signaling, and cooperate with mutant-β-catenin in thedevelopment of HCC (Tao J, Zhang R, Singh S, Poddar M, Xu E, Oertel M,Chen X, Ganesh S, Abrams M, Monga SP: Targeting beta-catenin inhepatocellular cancers induced by coexpression of mutant beta-cateninand K-Ras in mice. Hepatology 2016, the levels of both total and p-ERKwere assessed. Both total and p-ERK1 and total and p-ERK2 were notablydecreased following GC-1 treatment (FIG. 15B). p-AKT levels showedcomparable levels in the two groups (FIG. 15C). There was however, astriking decrease in p-STAT3 levels following GC-1 treatment (FIG. 15C).Thus GC-1 treatment led to a notable decrease in Met-ERK and Met-Stat3signaling to affect overall tumor burden in the hMet-S45Y-β-cateninmice.

Example 19 Ten-Day Treatment with GC-1 Decreases Tumor Burden inhMet-S45Y-β-Catenin HCC Model by Decreasing Tumor Cell Proliferation

To further validate the mechanism of GC-1 on tumorigenesis in thehMet-S45Y-β-catenin mouse model of HCC, a short-term GC-1 treatment wasperformed as described in methods and shown in FIG. 16A. Hepaticexpression of Diol was significantly increased in the 10-day GC-1 versuscontrol group (FIG. 16B).

To address effect of GC-1 on tumor burden, LWBW was compared between thetwo groups. A significant decrease (p=0.0022) in LWBW was evident afterGC-1 treatment (FIG. 16C). Grossly, the livers from basal diet and GC-1diet groups looked indistinguishable and without gross tumor nodules(FIG. 16D). H&E staining of liver sections showed several smallwell-differentiated tumor foci composed of cells with basophiliccytoplasm and some nuclear atypia (FIG. 17A). Staining for Myc-tagconfirmed presence of several tumor foci spread throughout liversections (FIG. 17A). Smaller and fewer tumor foci were apparent in theGC-1 diet fed group as observed by H&E and Myc-tag staining, althoughhistological features of tumors were not altered (FIG. 17A).

To address the basis of reduced tumor burden, the livers from bothgroups were compared for TUNEL. Comparable numbers of TUNEL-positivecells were evident between the two groups (FIG. 17B). No differences inthe number of CD45-positive between the two groups were observedsuggesting that intra- and extra-tumoral inflammation is unaffected byGC-1 (FIG. 17C).

Any effect of 10-day treatment of GC-1 on cell proliferation wasassessed by IHC for BrdU and Ki-67. A notable decrease in both markerswas evident within the tumor foci in the GC-1-treated group (FIG.18A-18B). Quantification revealed a significant decrease in thepercentage of Ki-67-positive cells within tumor nodules after GC-1treatment (FIG. 18C). No differences in Ki-67-positive cell numbers wereobserved in non-tumor areas between basal- and GC-diet (FIG. 18C).

Example 20 Ten-Day Treatment with GC-1 in hMet-S45Y-β-Catenin HCC Modeldoes not Impact Wnt Signaling

To investigate if there was any effect of short term GC-1 treatment onthe Wnt/β-catenin signaling, IHC was performed for cyclin-D1 and GS.Tumor nodules in basal diet group were strongly and uniformly positivefor cyclin-D1 as well as GS (FIGS. 19A-19B). After GC-1 treatment for 10days, tumors continued to be positive for cyclin-D1 as well as GSalthough a notable diminution in number and size of tumor foci wasvisible (FIGS. 19A-19B). Both GS and cyclin-D1 were predominantlylocalized the tumor foci.

Protein expression of total and active β-catenin was examined, alongwith cyclin-D1 and GS levels, by WB using the liver lysates from bothgroups. A modest decrease in the levels of total but not activeβ-catenin were observed in 10 days GC-1-diet groups (FIG. 19C).Similarly, marginal decreases in cyclin-D1 and modest decreases in GSlevels by WB were evident in the GC-1 group (FIG. 19C).

IHC and WB results together suggest that the seeming reduction in theWnt signaling following GC-1 may be actually be the result of, and notthe cause of, overall decreased tumor burden, because cyclin-D1 and GScontinued to be mostly expressed in the tumor foci in both control andtreatment groups. Thus, short term GC-1-treatment also did not impactWnt/β-catenin signaling and did not induce it to promote HCC burden.

Example 21 Ten-Day Treatment with GC-1 in hMet-S45Y-β-Catenin HCC ModelImpairs Met-Erk and Met-Stat3 Signaling

To further validate the impact on Met-Erk and Met-Stat3 signaling, liverlysates from 10-day GC-1 treated or control group were assessed. TotalMet levels were modestly decreased after GC-1 treatment (FIG. 20A). Aprofound decrease by in p-Met (Tyr 1234/1235) was observed in theGC-1-treated group and validated by two independent antibodies (FIG.20B). Likewise, while total ERK levels were unaffected, both p-ERK1 andp-ERK2 were dramatically decreased following GC-1 treatment as comparedto the controls (FIG. 20C). GC-1 treatment did not have any effect onp-AKT levels, however a profound decrease in p-STAT3 was evident (FIG.20C). Thus, GC-1 affects Met-ERK and Met-Stat signaling to reduce tumorburden in the hMet-S45Y-β-catenin mice.

There is a major unmet clinical need in the field of hepaticregenerative medicine as there are limited options for acute or chronichepatic insufficiency which will ultimately progress to end stage liverdisease. Treatment of end stage liver disease is often livertransplantation, however, a shortage of organs for transplantationnecessitates further methods (Collin de l'Hortet et al., AmericanJournal of transplantation 2016, 16:1688-96). T3 and GC-1 induce cellproliferation through upregulation of cyclin-D1 (Ledda-Columbano et al.,FASEB J 2006, 20:87-94; Pibiri et al., FASEB J 2001, 15:1006-13) whichin the liver, depends on Wnt/β-catenin signaling and PKA-dependentβ-catenin activation (Alvarado et al., Gene Expr 2016, 17:19-34; Fantiet al., Hepatology 2014, 59:2309-20). Also, β-catenin signaling isnormally activated during regeneration after acetaminophen-inducedhepatic injury or after partial hepatectomy, and β-catenin stabilizationitself promotes liver regeneration (Apte et al., Am J Pathol 2009,175:1056-65; Bhushan et al., Developmental biology 2008, 320:161-74;Monga, Gene Expr 2014, 16:51-62, Nejak-Bowen et al., Hepatology 2010,51:1603-13). GC-1 pretreatment before partial hepatectomy acceleratedcyclin-D1 expression after the surgery and led to an earlier transitionof hepatocytes into S-phase during liver regeneration (Alvarado et al.,Gene Expr 2016, 17:19-34).

Chronic hepatic injuries can benefit from regenerative therapies tosustain and even expand the residual functional hepatocyte mass.However, chronic hepatic injuries like viral hepatitis, non-alcoholic oralcoholic hepatitis and others, often and over an extended time period,lead to progressive fibrosis with regenerative nodules that maintainliver function and often complicated by acute-on-chronic liver failure(Sarin et al., Nature reviews Gastroenterology & hepatology 2016,13:131-49). As injury progresses and cirrhosis ensues, the risk fordevelopment of HCC also increases. Thus, it is pertinent to directlyaddress the effect of a regenerative therapy on HCC to especiallydemonstrate that it does not worsen the growth and development ofhepatic tumors. This is relevant for GC-1, which induces activation ofβ-catenin in normal liver to induce regeneration (Alvarado et al., GeneExpr 2016, 17:19-34). β-catenin activation due to mutations in CTNNB1 isa common event in HCC (Columbano et al., Endocrinology 2006,147:3211-8).

In vitro studies using three different cell lines with varying status ofβ-catenin gene mutations and associated Wnt/J-catenin activity, showedno effect of GC-1 on β-catenin-TCF4 activity. Furthermore, GC-1 neverincreased TopFlash activity in any of the liver tumor lines irrespectiveof CTNNB1 mutational status. While basal luciferase activity in thethree cell lines used was commensurate with CTNNB1 mutational status,such that HepG2 cells (exon-3 deletion) had the highest, Snu-398 cells(missense mutation in exon-3) the next highest, and Hep3B cells(wild-type) the lowest, GC-1 treatment did not alter the respectivebasal β-catenin-TCF4 activity.

To validate lack of effect of GC-1 on β-catenin-TCF4 activity and toinvestigate any effect of GC-1 on tumor growth and development in vivo,a clinically relevant model was utilized that represents 10% of humanHCC (Tao et al., Hepatology 2016, 64:1587-605). Stable expression ofmutant β-catenin and hMet in a subset of hepatocytes in mice leads toHCC, which partially β-catenin-addicted. Administration of GC-1 for 10or 21 days to these mice, once tumors were established, did not promoteWnt/β-catenin signaling in hepatic as shown by unaltered levels oftotal- and active-(hypophosphorylated) β-catenin along with its targetsGS and cyclin-D1. In fact, no difference in the localization orintensity of cyclin-D1 or GS within existing tumor nodules was observedafter GC-1 treatment as compared to the controls fed normal diet. Thiswas in contrast to the lipid nanoparticles used to deliver siRNA againstCTNNB1, which suppressed β-catenin expression, inhibited downstreamsignaling as demonstrated by the lack of GS and cyclin-D1 in tumornodules, and dramatically inhibited tumor growth in a related HCC model.Thus, GC-1 did not alter (did not increase) β-catenin activity in HCC invivo in the hMet-β-catenin model (FIG. 21).

Despite the lack of effect on Wnt signaling, GC-1 treatment decreasedoverall HCC burden in this model. This was desirable, as GC-1 use couldnot only induce regeneration but could affect tumorigenesis in chronicliver diseases (FIG. 21). Indeed, despite its mitogenic capacity, T3 waspreviously shown to accelerate remodeling of chemically-inducedpreneoplastic lesions in rats subjected to the resistant-hepatocytemodel of hepatocarcinogenesis (Ledda-Columbano et al., Cancer Res 2000,60:603-9). GC-1 was shown to also negatively influence the carcinogenicprocess through an induction of a differentiation program withinpreneoplastic hepatocytes based on analysis of molecular markers (Perraet al., Hepatology 2009, 49:1287-96). In the study disclosed herein, thepreviously unrecognized effect of GC-1 on inhibiting Met phosphorylationwas identified. Both at 10 days and 21 days, GC-1 treatment profoundlysuppressed Met-phosphorylation and modestly decreased total Met levels.Met phosphorylation and activation in the hMet-β-catenin model is due toMet overexpression, which leads to activation of Met signaling viaautophosphorylation at Y1234/1235. GC-1 inhibited Met phosphorylation atthese residues, which in turn impacted p-ERK1 and p-ERK2 as well asp-STAT3, without impacting P-AKT (FIG. 21).

The studies demonstrated that the use of GC-1 in chronic liver diseasesto induce regeneration was safe, and could be advantageous due to thetumor inhibitory effect.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method for increasing hepatocyte cell number and mass ina liver of a liver transplant donor, comprising selecting a subject hasdonated a portion of their liver; and administering to the subject aneffective amount of a pharmaceutical composition comprising GC-1(sobetirome) or a pharmaceutically acceptable salt thereof, wherein thepharmaceutical composition is administered to the subject within one dayafter a resection of the portion of their liver, and is continued for atmost three weeks following the resection of the portion of their liver;thereby increasing hepatocyte cell number and mass in the liver of thesubject.
 2. The method of claim 1, wherein the subject is a high riskdonor.
 3. The method of claim 1, wherein the subject is older than about45 years of age.
 4. The method of claim 1, wherein the GC-1 or thepharmaceutically acceptable salt thereof is administered intravenouslyto the subject.
 5. The method of claim 4, wherein the GC-1 or thepharmaceutically acceptable salt thereof is administered at a dose of0.1 mg/kg to about 0.5 mg/kg.
 6. The method of claim 5, the GC-1 or thepharmaceutically acceptable salt thereof is administered at a dose ofabout 0.3 mg/kg.
 7. The method of claim 1, wherein the GC-1 or thepharmaceutically acceptable salt thereof is administered orally to thesubject.
 8. The method of claim 1, wherein the GC-1 or thepharmaceutically acceptable salt thereof is administered for at most 14days after liver transplantation.
 9. The method of claim 1, furthercomprising monitoring the metabolic function of the liver in thesubject.
 10. The method of claim 1, further comprising measuring liversize in the subject.
 11. The method of claim 1, further comprisingobtaining a lipid profile of the subject.
 12. The method of claim 1,wherein the subject is human.
 13. The method of claim 1, wherein theGC-1 or the pharmaceutically acceptable salt thereof is administeredwithin one day of the donation of their liver.
 14. The method of claim1, wherein the GC-1 or the pharmaceutically acceptable salt thereof isadministered orally, and the subject is human.
 15. The method of claim14, wherein 10 μg to 100 μg of GC-1 or the a pharmaceutically acceptablesalt thereof is administered once, twice or three times per day.
 16. Themethod of claim 1, wherein the GC-1 or the pharmaceutically acceptablesalt thereof is administered for seven to fourteen days following theresection of the liver in the subject.
 17. The method of claim 1,wherein hepatocyte proliferation is measured by radiological monitoringof hepatic size in the subject.
 18. The method of claim 1, furthercomprising measuring an increase in hepatocyte cell number in thesubject.
 19. A method for increasing hepatocyte cell number and mass ina liver of a subject that has hepatoxicity due to an acetaminophenoverdose, comprising selecting the subject that has hepatoxicity due toan acetaminophen overdose, and administering to the subject that hashepatoxicity due to the acetaminophen overdose an effective amount of apharmaceutical composition comprising GC-1 (sobetirome) or apharmaceutically acceptable salt thereof, wherein the pharmaceuticalcomposition is administered to the subject within 72 hours of theacetaminophen overdose; thereby increasing hepatocyte cell number andmass in the liver of the subject.
 20. A method for increasing hepatocytecell number and mass in a liver transplant in a subject, comprisingselecting a subject that is the recipient of a liver transplant, whereinthe subject has small for size syndrome (SFSS); administering to thesubject an effective amount of a pharmaceutical composition comprisingGC-1 (sobetirome) or a pharmaceutically acceptable salt thereof, whereinthe pharmaceutical composition is administered to the subject within oneday after liver transplantation and is continued for at most three weeksfollowing the liver transplantation; thereby increasing hepatocyte cellnumber and mass in the liver transplant in the subject.